Functionalized and crosslinked polymers

ABSTRACT

Polyhydric polymers may be converted to derivatives thereof by reaction with divinyl sulfone to provide vinyl sulfone substituted polymers, where the polymers may additionally be further derivatized, including crosslinked, and the crosslinked and non-crosslinked derivatives may be used in biomedical and other applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 62/553,371 filed Sep. 1, 2017, whichapplication is incorporated herein by reference in its entirety for allpurposes.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to functionalized polymersincluding crosslinked versions thereof, and methods for preparation anduses thereof. The polymers of the present disclosure provide usefulproperties that are not available from polymers currently available.

BACKGROUND

The use of polymeric materials as biomaterials has grown in recentyears, resulting in an expanding polymeric toolbox. Both synthetic andnatural polymers have been used as components for biomaterials, andtheir unique chemical structures can provide specific functions fordesired applications. The use of polymers as biomaterials has expandedbecause of advances in the synthesis of polymers with controlled andfunctional architectures, which has improved the range of materialspossible, as well as their biocompatibility. While these biocompatiblepolymers are useful because they do not specifically interact withbiological systems, this has also hindered their use in applications inwhich natural physiological interactions are desired to manipulatebiological responses such as wound healing, cellular or growth factorbinding, or enzymatic degradation. Thus, there is interest in usingpolymers, such as those found in living organisms, that are modified toprovide properties that are improved or different from those ofunmodified polymers. An example of such polymers are those comprisingpolyhydric alcohols. A non-limiting example of such a polymer ishyaluronic acid (HA) polymer.

HA is a non-sulphated glycosaminoglycan (GAG) and is composed ofrepeating polymeric disaccharides of D-glucuronic acid andN-acetyl-D-glucosamine linked by a glucuronidic β(1→3) bond. In aqueoussolutions, HA forms specific stable tertiary structures. Despite thesimplicity in its composition, without variations in its sugarcomposition or without branching points, HA has a variety ofphysicochemical properties. HA polymers occur in a number ofconfigurations and shapes, depending on their size, salt concentration,pH, and associated cations. Unlike other GAG, in living organisms, HA isnot covalently attached to a protein core, but it may form aggregateswith proteoglycans. HA encompasses a large volume of water givingsolutions high viscosity, even at low concentrations.

HA is involved in multiple physiological processes, for example, skin. Akey molecule involved in skin moisture is hyaluronan or hyaluronic acid(HA), a glycosaminoglycan (GAG) with a unique capacity to bind andretain water molecules. HA belongs to the extracellular matrix (ECM)molecules. ECM molecules that lie between cells, in addition toproviding a constructive framework, exert major effects on cellularfunction. These ECM molecules, although they appear amorphous by lightmicroscopy, they form a highly organized structure, comprising mainly ofGAG, proteoglycans, growth factors and structural proteins such ascollagens, with the predominant component of the skin ECM is HA.

What is needed are derivatives of polyhydric polymers, compositions ofsuch polymers and use of such polymers for medical treatments andproduction of medical devices and components.

SUMMARY

In brief, the present disclosure provides polymers, method of makingpolymers, and methods of using polymers.

-   1) For example, in one aspect, the present disclosure provides a    derivative of a polyhydric polymer, such as a polysaccharide, e.g.,    polyhyaluronic acid, in which one or more hydroxyl groups of the    hyaluronic acid is a modified hydroxyl group, wherein the derivative    of hyaluronic acid or other polyhydric polymer has the structure    HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) where HA is a polyhydric polymer    such as hyaluronic acid, X is S or NH, R₁ is a substituted or    unsubstituted C₁-C₂₀ aliphatic or aromatic moiety and n is the    number of modified hydroxyl groups where n is an integer and n≥1,    and Y is one or more of H, a carboxylic acid group or a salt or    ester thereof, a hydroxyl group, a sulfonic acid group or a salt    thereof, or an amine group.

In another aspect, the present disclosure provides a derivative of apolyhydric polymer, such as a polysaccharide, e.g., hyaluronic acid, inwhich two or more hydroxyl groups of the hyaluronic acid are modifiedhydroxyl groups, wherein the derivative of hyaluronic acid or otherpolyhydric polymer has the structure(Y—R₂—X—CH₂CH₂SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) where HAis hyaluronic acid or other polyhydric polymer, X is S or NH, R₁ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, R₂ isa substituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moietywherein R₁ and R₂ are different from each other, wherein n and m areeach integers, and n≥1 and m≥1, and Y is H; a carboxylic acid group, ora salt or ester; thereof; a hydroxyl group; a sulfonic acid group, or asalt thereof, or an amine group.

In another aspect, the present disclosure provides a derivative ofpolyhydric polymer, such as a polysaccharide, e.g., hyaluronic acid, inwhich two or more hydroxyl groups of the hyaluronic acid are modifiedhydroxyl groups, wherein the derivative of hyaluronic acid has thestructure (CH₂═CH—SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) whereHA is hyaluronic acid or other polyhydric polymer, X is S or NH, R₁ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, eachof n and m is an integer, and n≥1 and m≥1, and Y is H; a carboxylic acidgroup, or a salt or ester; thereof; a hydroxyl group; a sulfonic acidgroup, or a salt thereof; or an amine group.

In further aspects, the present disclosure provides derivatives ofpolyhydric polymers such as described above, which are furthercharacterized by wherein 0.25-50% of a sum of the hydroxyl groups andthe modified hydroxyl groups are a modified hydroxyl group.

In another aspect, the present disclosure comprises crosslinked polymerscomprising a reaction product of a derivative of a polyhydric polymerdisclosed herein, such as a polysaccharide, e.g., hyaluronic acid, andoptionally, may comprise a crosslinking agent, wherein as used herein, acrosslinking agent may comprise known crosslinking agents, such ascrosslinking compounds, for example, OH crosslinking agents or vinylcrosslinking agents, FeCl₃, or compounds and/or energy sources,including but not limited to, UV and related photoinitiator compounds.

In an aspect, the present disclosure comprises crosslinked polymerscomprising a reaction product of a derivative of a polyhydric polymerdisclosed herein, such as a polysaccharide, e.g., hyaluronic acid, andoptionally, may comprise a crosslinking agent, wherein

a) the derivative of the polyhydric polymer, such as hyaluronic acid hasthe structure HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) wherein one or morehydroxyl groups of the hyaluronic acid is a modified hydroxyl group, andwherein HA is the polyhydric polymer, e.g., hyaluronic acid, comprisinghydroxyl groups, X is S or NH, R¹ is a substituted or unsubstitutedC₁-C₂₀ aliphatic or aromatic moiety and n is the number of modifiedhydroxyl groups where n≥1, and Y is H; a carboxylic acid group, or asalt or ester; thereof; a hydroxyl group; a sulfonic acid group, or asalt thereof; or an amine group; and

b) the crosslinking agent comprises at least two functional groups thatare capable of reacting with the hydroxyl groups of the derivative ofhyaluronic acid.

In another aspect, the present disclosure provides a crosslinked polymercomprising a reaction product of a derivative of a polyhydric polymerdisclosed herein, such as a polysaccharide, e.g., hyaluronic acid, and acrosslinking agent, wherein

a) the derivative of a polyhydric polymer, comprises two or morehydroxyl groups of the hyaluronic acid as modified hydroxyl groups,wherein the derivative of hyaluronic acid or other polyhydric polymerhas the structure(Y—R₂—X—CH₂CH₂SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) where HAis hyaluronic acid or other polyhydric polymer, X is S or NH, R₁ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, R₂ isa substituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moietywherein R₁ and R₂ are different from each other, wherein n and m areeach integers, and n≥1 and m≥1, and Y is H; a carboxylic acid group, ora salt or ester; thereof; a hydroxyl group; a sulfonic acid group, or asalt thereof; or an amine group; and

b) the crosslinking agent comprises at least two functional groups thatare capable of reacting with the hydroxyl groups of the derivative ofhyaluronic acid.

In another aspect, the present disclosure provides crosslinked polymerscomprising a reaction product of a derivative of a polyhydric polymer,such as a polysaccharide, e.g., hyaluronic acid, disclosed herein, and acrosslinking agent, wherein

a) the derivative of hyaluronic acid comprises vinyl groups and has thestructure (CH₂═CH—SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y)_(n)wherein two or more hydroxyl groups of the hyaluronic acid are modifiedhydroxyl groups, HA is hyaluronic acid comprising hydroxyl groups, X isS or NH, R¹ is a substituted or unsubstituted C₁-C₂₀ aliphatic oraromatic moiety, n≥1 and m≥1, and Y is H; a carboxylic acid group, or asalt or ester; thereof; a hydroxyl group; a sulfonic acid group, or asalt thereof; or an amine group.; and

b) the crosslinking agent comprises at least two functional groups thatare capable of reacting with the vinyl groups of the derivative ofhyaluronic acid or

c) the crosslinking agent comprises at least two functional groups thatare capable of reacting with the hydroxyl groups of the derivative ofhyaluronic acid or

d) The crosslinking agent comprises functional groups that are capableof ionic crosslinking or

e) the crosslinking agent comprises functional groups that are capableof thermal crosslinking or

f) the crosslinking agent generates free radicals that are capable offree radical crosslinking

In further aspects, a crosslinked polymer disclosed herein may befurther characterized by wherein 0.25-50% of a sum of the hydroxylgroups and the modified hydroxyl groups are a modified hydroxyl group.

In another aspect, the present disclosure provides a process comprising:

a) reacting hydroxyl groups attached to a polymer, such as hydroxylgroups on hyaluronic acid (HA) or other polyhydric polymer, with divinylsulfone (DVS) to provide a first derivative of the polymer; and

b) reacting the first derivative of the polymer with a nucleophile of aformula selected from X′—R¹—Y and X′—R²—Y to provide a second derivativeof the polymer;

wherein R¹ is a substituted or unsubstituted C₁-C₂₀ aliphatic oraromatic moiety, R² is a substituted or unsubstituted C₁-C₂₀ aliphaticor aromatic moiety, X′ is a nucleophilic group, comprising a thiol oramine, and Y is one or more of H, a carboxylic acid group or a salt orester thereof, a hydroxyl group, a sulfonic acid group or a saltthereof, or an amine group. A polymer that has undergone one or morederivatization reactions includes, and is referred to herein as, aderivative of a polyhydric polymer, whether the polymer has beenderivatized one or more times, e.g., one, two, three, four, etc.derivatizations of the initial polymer. A derivative of a polymer mayalso be referred to as a first derivative, a second derivative, a thirdderivative, etc., of a polymer.

In further aspects, the process may be further characterized by one ormore of the following:

1) The process wherein 0.25-50% of the hydroxyl groups present on theinitial (underivatized) polymer are converted to oxyethyl ethenylsulfone groups of the formula —OCH₂CH₂—SO₂CH═CH ₂.

2) The process wherein the polymer is hyaluronic acid and the firstderivative of the polymer is an oxyethyl ethenyl sulfone derivative ofthe hyaluronic acid is HA-(—OCH₂CH₂SO₂CH═CH₂)_(n) (HA-DVS).

3) The process wherein the second derivative isHA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹)n (HA-DVS-N).

4) The process wherein 0.25-50% of the hydroxyl groups present on thepolymer are converted to —OCH₂CH₂SO₂CH₂CH₂—X—R¹ groups.

5) The process wherein X′ is thiol or amine and X is —S— or —NH—.

6) The process wherein the second derivative isHA-(—OCH₂CH₂SO₂CH₂CH₂—X—R²—Y)_(n) (HA-DVS-NY).

7) The process of wherein 0.25-50% of the hydroxyl groups present on thepolymer are converted to —OCH₂CH₂SO₂CH₂CH₂—X—R²—Y groups as calculatedby the sum of the hydroxyl groups and the modified hydroxyl groups.

8) The process wherein X is —S—.

9) The process wherein Y is hydroxyl.

10) The process wherein Y is carboxylic acid or a salt or ester thereof.

11) The process wherein Y is sulfonic acid of a salt of ester thereof.

12) The process further comprising reacting the second derivative of thepolymer with a crosslinking agent to provide a third derivative of thepolymer, where the third derivative is a crosslinked polymer.

In another aspect, the present disclosure provides a derivative of apolyhydric polymer, such as a derivative of hyaluronic acid, prepared byany of the processes identified herein. In another aspect, the presentdisclosure provides a crosslinked polymer prepared by any of theprocesses identified herein.

In another aspect, the present disclosure provides a compositioncomprising a derivative of a polymeric polyhydric alcohol, e.g.,hyaluronic acid, wherein derivatives of a polymeric polyhydric alcoholmay comprise the derivatives as disclosed above for polyhydric polymers.For example, HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n);(Y—R₂—X—CH₂CH₂SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n);(CH₂═CH—SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y)_(n); or(CH₂═CH—SO₂CH₂CH₂O)_(m)-HA, where HA is hyaluronic acid or otherpolyhydric polymer, X is S or NH, R₁ is a substituted or unsubstitutedC₁-C₂₀ aliphatic or aromatic moiety, R₂ is a substituted orunsubstituted C₁-C₂₀ aliphatic or aromatic moiety, and where applicable,wherein R₁ and R₂ are different from each other, Y is one or more of H,a carboxylic acid group or a salt or ester thereof, a hydroxyl group, asulfonic acid group or a salt thereof, or an amine group; wherein n andm are each integers, and n≥1 and m≥1 A composition may further comprisean excipient.

In another aspect, the present disclosure provides a compositioncomprising a crosslinked polymer, e.g., a crosslinked derivative ofpolymeric hyaluronic acid, as described herein. A composition mayfurther comprise an excipient. Each of the compositions disclosed hereinmay optionally include one or more of a pharmaceutically acceptablesynthetic polymer, thermosreversible polymer, biodegradable polymer,buffer, complexing agent, tonicity modulator, ionic strength modifier,solvent, anti-oxidant, preservative, viscosity modifier, pH modifier,surfactant, emulsifier, phospholipid, stabilizer and porogen. Alsooptionally, a composition as disclosed herein may further comprise abiologically active agent.

Derivatized polymers and compositions comprising one or more derivatizedpolymers disclosed herein exhibit shear thinning. Shear thinning is thenon-Newtonioan behavior of fluids whose viscosity decreases under shearstrain.

In additional aspects, the present disclosure provides method of usingthe polymers and compositions as disclosed herein. For example, thepresent disclosure provides the following aspects:

1) A wound healing device comprising a composition as described herein.

2) A method for wound healing comprising administering to a subject inneed thereof an effective amount of a composition as described herein.

3) A bulking agent comprising a composition as described herein.

4) A dermal filler comprising a composition as described herein.

5) A method of filling a void in a subject in need thereof comprisingadministering to the subject a dermal filler as described herein.

6) A viscosupplement comprising a composition as described herein.

7) A method of relieving joint pain in a subject in need thereof,comprising administering to the subject a viscosupplement as describedherein.

8) A method of preventing surgical adhesions in a subject in needthereof comprising administering the subject an effective amount of acomposition as described herein.

9) A tissue sealant comprising a composition as described herein.

10) A method of sealing tissue in a subject in need thereof comprisingadministering to the subject an effective amount of a tissue sealant asdescribed herein.

11) A method of treating bacterial vaginosis in a subject in needthereof comprising administering to the subject an effective amount of acomposition as described herein.

12) A nasal treatment device comprising a composition as describedherein.

13) A method of treating a nasal condition in a subject in need thereofcomprising administering the subject an effective amount of acomposition as described herein.

14) An eye drop comprising a composition as described herein.

15) A method of treating an ocular condition in a subject in needthereof comprising administering the subject an effective amount of acomposition as described herein.

16) A punctal plug comprising a composition as described herein.

17) A method of treating mucocitis in a subject in need thereofcomprising administering to the subject an effective amount of acomposition as described herein.

18) An anti-bacterial formulation comprising a composition as describedherein.

19) An ear treatment device comprising a composition as describedherein.

20) A method of treating an ear condition comprising administering to asubject in need thereof an effective amount of a composition asdescribed herein.

21) A method of drug delivery to a subject in need thereof comprisingadministering to the subject an effective amount of a composition asdescribed herein that comprises the drug.

22) A biopsy plug comprising a composition as described herein.

23) A plug for female sterilization comprising a composition asdescribed herein.

24) A method of female sterilization to a subject in need thereofcomprising administering to the subject an effective amount of acomposition as described herein.

25) A tissue scaffold comprising a composition as described herein.

26) The method of supporting tissue growth in a subject in need thereofcomprising implanting in the subject a tissue scaffold as describedherein.

27) A burr hole plug comprising a composition as described herein.

28) A nerve guide comprising a composition as described herein.

29) A vaginal lubricant comprising a composition as described herein.

30) A coating for a device comprising a composition as described herein.

31) A method for coating a device comprising applying a coating asdescribed herein onto a surface of the device.

32) A method of administering an injectable formulation comprising acomposition as described herein.

33) A method for additive manufacturing comprising a polymer asdescribed herein, e.g., a derivative of hyaluronic acid as describedherein, or prepared by a process as described herein, to provide aderivative of the polymer, e.g., hyaluronic acid, and depositing thederivative onto a substrate to provide an article formed by additivemanufacturing.

34) A method for additive manufacturing by deposition of a with apolymer as described herein, e.g., a derivative of hyaluronic acid asdescribed herein on the surface or within a polymeric substrate.

35) A method of coating or penetrating an article formed by additivemanufacturing with a polymer as described herein, e.g., a derivative ofhyaluronic acid as described herein.

36) An electrospun material or article comprising a composition asdescribed herein.

37) A method for producing an electrospun material or article,comprising producing, with an electrospinning device, a material or anarticle comprising a derivative of hyaluronic acid described herein.

38) A method of coating or penetrating an article formed byelectrospinning with a polymer as described herein, e.g., a derivativeof hyaluronic acid as described herein.

39) A textile material or article comprising a composition as describedherein.

40) A method for producing a textile material or article, comprisingproducing, with an electrospinning device, a material or an articlecomprising a derivative of hyaluronic acid described herein.

41) A method of coating or penetrating an article formed byelectrospinning on a textile substrate with a polymer as describedherein, e.g., a derivative of hyaluronic acid as described herein.

The above-mentioned and additional features of the present disclosureand the manner of obtaining them will become apparent, and thedisclosure will be best understood by reference to the following moredetailed description. All references disclosed herein are herebyincorporated by reference in their entirety as if each was incorporatedindividually.

This Brief Summary has been provided to introduce certain concepts in asimplified form that are further described in detail below in theDetailed Description. Except where otherwise expressly stated, thisBrief Summary is not intended to identify key or essential features ofthe claimed subject matter, nor is it intended to limit the scope of theclaimed subject matter.

The details of one or more aspects are set forth in the descriptionbelow. The features illustrated or described in connection with oneexemplary aspect may be combined with the features of other aspects.Thus, any of the various aspects described herein can be combined toprovide further aspects. Aspects of the aspects can be modified, ifnecessary to employ concepts of the various patents, applications andpublications as identified herein to provide yet further aspects. Otherfeatures, objects and advantages will be apparent from the description,the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary features of the present disclosure, its nature and variousadvantages will be apparent from the accompanying drawings and thefollowing detailed description of various aspects. Non-limiting andnon-exhaustive aspects are described with reference to the accompanyingdrawings, wherein like labels or reference numbers refer to like partsthroughout the various views unless otherwise specified. The sizes andrelative positions of elements in the drawings are not necessarily drawnto scale. For example, the shapes of various elements are selected,enlarged, and positioned to improve drawing legibility. The particularshapes of the elements as drawn have been selected for ease ofrecognition in the drawings. One or more aspects are describedhereinafter with reference to the accompanying drawings in which:

FIG. 1 shows a ¹H NMR spectrum of a divinyl sulfone-modified hyaluronicacid according to the present disclosure.

FIG. 2 shows exemplary reactions of the present disclosure.

FIG. 3 shows exemplary reactions of the present disclosure.

FIG. 4 shows a ¹H-NMR spectrum of a 2-mercaptobenzoic acid (MBA)modified hyaluronic acid according to the present disclosure.

FIG. 5 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 6 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 7 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 8 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 9 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 10 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 11 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 12 is a graph showing a characteristic of an exemplary derivatizedpolymer disclosed herein.

FIG. 13 is a graph showing cellular growth on exemplary derivatizedpolymers disclosed herein.

FIG. 14 is a graph showing cellular growth on electrospun articlescomprising exemplary derivatized polymers disclosed herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure may be understood more readily by reference tothe following detailed description of preferred aspects of thedisclosure and the Examples included herein.

In one aspect, the present disclosure provides functionalized polymersincluding crosslinking versions thereof. A functionalized polymer refersto an organic polymer comprising hydroxyl groups, and optionally alsoincluding a second functional group, such as a carboxylic acid, amine orsulfonic acid group.

The present disclosure provides a derivative of a polyhydric polymer,such as a polysaccharide, e.g., hyaluronic acid, in which one or morehydroxyl groups of the hyaluronic acid is a modified hydroxyl group,wherein the derivative of hyaluronic acid or other polyhydric polymerhas the structure HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) where HA is apolyhydric polymer such as hyaluronic acid, X is S or NH, R₁ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety and nis the number of modified hydroxyl groups where n is an integer and n≥1,and Y is one or more of H, a carboxylic acid group or a salt or esterthereof, a hydroxyl group, a sulfonic acid group or a salt thereof, aphosphonic acid group or a salt thereof, or an amine group.

In another aspect, the present disclosure provides a derivative of apolyhydric polymer, such as a polysaccharide, e.g., hyaluronic acid, inwhich two or more hydroxyl groups of the hyaluronic acid are modifiedhydroxyl groups, wherein the derivative of hyaluronic acid or otherpolyhydric polymer has the structure(Y—R₂—X—CH₂CH₂SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) where HAis hyaluronic acid or other polyhydric polymer, X is S or NH, R₁ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, R₂ isa substituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moietywherein R₁ and R₂ are different from each other, wherein n and m areeach integers, and n≥1 and m≥1, and Y is H; a carboxylic acid group, ora salt or ester; thereof; a hydroxyl group; a sulfonic acid group or asalt thereof; a phosphonic acid group or a salt thereof; or an aminegroup.

In another aspect, the present disclosure provides a derivative ofpolyhydric polymer, such as a polysaccharide, e.g., hyaluronic acid, inwhich two or more hydroxyl groups of the hyaluronic acid are modifiedhydroxyl groups, wherein the derivative of hyaluronic acid has thestructure (CH₂═CH—SO₂CH₂CH₂O)m-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n) where HAis hyaluronic acid or other polyhydric polymer, X is S or NH, R₁ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, eachof n and m is an integer, and n≥1 and m≥1, and Y is H; a carboxylic acidgroup, or a salt or ester; thereof; a hydroxyl group; a sulfonic acidgroup, or a salt thereof; a phosphonic acid group or a salt thereof; oran amine group.

In further aspects, the present disclosure provides derivatives ofpolyhydric polymers such as described above, which are furthercharacterized by the derivative wherein 0.25-50% of a sum of thehydroxyl groups and the modified hydroxyl groups are a modified hydroxylgroup.

In another aspect, the present disclosure comprises crosslinked polymerscomprising a reaction product of a derivative of a polyhydric polymerdisclosed herein, such as a polysaccharide, e.g., hyaluronic acid, andoptionally, may comprise a crosslinking agent, wherein as used herein, acrosslinking agent may comprise known crosslinking agents, such ascrosslinking compounds, for example, OH crosslinking agents or vinylcrosslinking agents, FeCl₃, or compounds and/or energy sources,including but not limited to, UV and related photoinitiator compounds.

In one aspect the present disclosure utilizes a polysaccharide with oneor more available hydroxyl groups and reacts one or more of thosehydroxyl groups under specific conditions as disclosed herein withdivinyl sulfone such that only one of the vinyl groups of the divinylsulfone reacts with the hydroxyl group via an addition reaction to forman ether bond between the polysaccharide and the residue of the divinylsulfone. The degree of reaction can range from about 0.5% to about 50%of the available hydroxyl groups. At higher substitution, i.e., around50%, some degree of crosslinking will typically occur. Thus, the presentdisclosure provides vinyl sulfone substituted polysaccharide polymerswith minimal to no crosslinking, or polysaccharide polymers that have alevel of vinyl sulfone substitution crosslinking due to double reactionof the divinyl sulfone (i.e., reaction of both ethenyl groups of the DVSwith hydroxyl groups).

The residual vinyl group of the vinyl sulfone can be then reacted with acompound that has a reactive thiol group. This reaction occurs via aMichael addition between the residual vinyl group of the divinyl sulfoneand the free thiol group such that a thioether bond is formed. There arenumerous variations of the degree of substitution, the thiol derivativeused, the sequence of the reactions and the replication of reactionsthat provide a large variety of derivatives of polymeric polyhydricalcohols and compositions contemplated and disclosed herein. Derivativesof polymeric polyhydric alcohols can be crosslinked in many differentways, and compositions comprising such crosslinked derivatives ofpolymeric polyhydric alcohols are contemplated and disclosed herein. Thederivatives of polymeric polyhydric alcohols and compositions thereofhave numerous medical and non-medical applications. Methods of use ortreatment disclosed herein may comprise derivatives of polymericpolyhydric alcohols and compositions thereof. Derivatives of polymericpolyhydric alcohols may also be referred to herein as polyhydric polymerderivatives.

The derivatives of polymeric polyhydric alcohols and compositionsthereof of the present disclosure are prepared as described herein.Typically, a polymer having hydroxyl groups is combined with divinylsulfone (DVS) under suitable reaction conditions. Those reactionconditions include a suitable pH of the solution, where the reactiontypically occurs under basic conditions, e.g., a pH of 11-14, or 12-13,e.g., about 12.3. The reaction conditions include a suitable solvent,where water or DMSO are suitable solvents, e.g., the reaction may beconducted in water. The description of reaction conditions may furtherinclude stirring the reacting mixture, e.g., stirring with a stirringrate of >200 rpm (rotations per minute), such as 250-800 rpm.Furthermore, the description of reaction conditions may includespecification of the relative amounts of DVS and polymer (e.g.,polysaccharide) that are combined, where these relative amounts may beexpressed in terms of moles of DVS to moles of repeat unit in thepolymer. For instance, the method for preparing the functionalizedpolymer may be described in terms of the ratio of DVS:polymer repeatunit, where this ratio may be at least 0.5:1, e.g., up to about 5:1, orup to about 7.5:1, or up to about 10:1, or up to about 15:1, or up toabout 20:1

An exemplary functionalized polymer is a polysaccharide, where anexemplary polysaccharide is hyaluronic acid (HA). HA is a polysaccharideillustrated by the structure shown below.

HA contains two different functional groups, namely hydroxyl groups andcarboxylic acid groups. HA also contains ether and acetamide groups,however these are essentially chemically inert. In commerciallyavailable preparations of HA, some or all of the carboxylic acids may bepresent as the corresponding salt, e.g., as the sodium, potassium orammonium salt. In the present disclosure, and unless the contextindicates otherwise, HA refers inclusively to polymers of the structureshown above as well as the corresponding carboxylate salts of thosepolymers. Another exemplary polysaccharide polymer is dextran. Otherexemplary polysaccharide polymers useful in the presentdisclosureinclude, but are not limited to, sodium alginate, calciumalginate, dextran, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methyl cellulose, hyaluronic acid,hyaluronic acid derivatives, dextran, heparin, chitosan, xantham gum,Xylan, guar gum, pullulan or locust bean gum. The term “polyhydricpolymer” includes, but is not limited to, such polymericpolysaccharides. The term “polyhydric polymer” includes, but is notlimited to polymers that have two or more hydroxyl groups, and may alsobe referred to herein as polymeric polyhydric alcohols

In one aspect, the present disclosure provides a process wherein apolymeric polysaccharide that has an available hydroxyl group, i.e., ahydroxyl group that is capable of undergoing a reaction with divinylsulfone, is reacted with DVS under basic conditions. If the conditionsare selected appropriately, the reaction can be controlled such that oneof the vinyl groups of the divinyl sulfone will react with a freehydroxyl group of the polysaccharide such that the polysaccharide doesnot crosslink to such an extent that it forms a hydrogel. This resultsin the polysaccharide being functionalized with the divinyl sulfone suchthat one of the vinyl groups undergoes reaction with a hydroxyl group ofthe polysaccharide and the other vinyl group remains functional. Thevinyl group of the divinyl sulfone reacts with the hydroxyl group by anaddition reaction that results in an ether linkage.

The reaction may be performed under basic conditions with a pH ofgreater than 11. Optionally, the pH is in the range of 12.0 to 13.5.Optionally, the pH is in the range is in the 12.0 to 12.5 range.Optionally, the pH range is in the 12.2 to 12.7 range.

To ensure that the predominant reaction is a single reaction of one ofthe vinyl groups of the divinyl sulfone, and not a crosslinking reactionin which predominantly both the vinyl groups react with hydroxyl groupsof the polysaccharide to form a crosslinked gel, the molar ratio of thedivinyl sulfone to that of the polysaccharide repeat units is greaterthan 1. In one aspect, the molar ratio of the divinyl sulfone to that ofthe polysaccharide repeat units is greater than 5. In one aspect, themolar ratio of the divinyl sulfone to that of the polysaccharide repeatunits is greater than 7. In one aspect, the molar ratio of the divinylsulfone to that of the polysaccharide repeat units is greater than 10.In one aspect, the molar ratio of the divinyl sulfone to that of thepolysaccharide repeat units is greater than 15. In an aspect, the molarratio of the divinyl sulfone to that of the polysaccharide repeat unitsis from about 1 to about 20, or from about 1 to about 15, or from about1 to about 10, or from about 1 to about 5, or from about 5 to about 20,or from about 5 to about 15, or from about 5 to about 10, of from about10 to about 20, or from about 10 to 15, or is about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

To provide intimate contact between the reactants, the reaction mixturemay be stirred. Methods and devices for adequate mixing are known tothose of skill in the art. For example, in order to ensure that there isadequate stirring of the reaction solution during the reaction, therotational speed of the mixing impellor should be controlled. In oneaspect, the revolutions per minute (rpm) of the mixing impellor shouldbe in the range of 200 to 400 rpm. In another aspect, the revolutionsper minute (rpm) of the mixing impellor should be should be in the rangeof 400 to 600 rpm. In another aspect, the revolutions per minute (rpm)of the mixing impellor should be should be in the range of 600 to 800rpm.

The amount of substitution accomplished may be controlled, in part, bythe duration of exposure of the polysaccharide to the divinyl sulfone ata pH of greater than 11 (reaction time). In one aspect, the reactiontime can range from 10 seconds through to 60 minutes. In one aspect, thereaction time can be in the range of 2 minutes to 35 minutes. In anotheraspect, the reaction time can be in the range of 4 minutes to 30minutes, or from 20 minutes to 60 minutes, from 15 minutes to 20minutes, from 5 minutes to 10 minutes, from 10 seconds to 30 seconds,from 30 second to 1.5 minutes, and ranges thereinbetween

The solvent that can be used for the reaction can be water, water withan ionic modifier, for example NaCl, a combination of water and awater-miscible solvent. Water miscible solvents can include but are notlimited to methanol, ethanol, isopropanol, dimethyl formamide (DMF),acetone, 1,4-dioxane, pyridine, dimethyl sulfoxide (DMSO),tetrahydrofuran (THF) and acetonitrile.

The temperature of the reaction mixture can also be used to influencethe amount of substitution of the polysaccharide by the divinyl sulfone.In one aspect, the reaction mixture can be maintained at a temperaturethat is lower than 25° C. so as to reduce the rate of the reaction. Thiscan enable lower substitution levels for the same duration as comparedto room temperature or it can allow for a longer reaction time that thatat room temperature to yield a similar amount of substitution. In oneaspect, the temperature can be in the 15° C. to 20° C. range. In anotheraspect, the reaction mixture can be in the 10° C. to 15° C. range. Inyet another aspect, the temperature can be in the 2° C. to 10° C. range.In another aspect, the temperature can be increase above 25° C. so as toprovide shorter reaction times as compared to 25° C. to get similaramounts of substitution or to get greater substitution as compared to25° C. for an equivalent amount of reaction time. In one aspect, thereaction mixture can be in the 28° C. to 35° C. range. In anotheraspect, the reaction mixture can be in the 36° C. to 50° C. range. Inanother aspect, the reaction mixture can be in the 51° C. to 75° C.range.

The amount of substitution, as measured by the molar ratio of theattached vinyl group from the divinyl sulfone to the polysacchariderepeat unit, can be greater than 5%. In one aspect, for polysaccharideswith at least one hydroxyl group, the amount of substitution is in therange of 5% to 35%. In another aspect, for polysaccharides with at leastone hydroxyl group, the amount of substitution is in the range of 36% to70% range. In another aspect, for polysaccharides with at least onehydroxyl group, the amount of substitution is in the range of 71% to100% range. In another aspect, for polysaccharides with at least twohydroxyl groups, the amount of substitution is in the range of 101% to200% range.

In one aspect, polysaccharide polymers that comprise at least onehydroxyl group that is available for reaction with divinyl sulfone underconditions where the pH is greater than 11 are suitable for use in thisdisclosure. Such polysaccharides include, but are not limited tohyaluronic acid and its sodium or potassium salts, hyaluronic acidderivatives, dextran and dextran derivatives, dextran sulfate, heparin,chitosan and derivatives thereof, xylan, guar gum, locust bean gum,chondroitin 6-sulfate, chondroitin 4-sulfate, heparan sulfate, keratinsulfate, dermatan sulfate and chitin. In an aspect, the polysaccharidepolymer is hyaluronic acid or sodium hyaluronate. In another aspect, thepolysaccharide is dextran. As used herein, polysaccharide meanspolymeric polysaccharide molecules.

The molecular weight of the polysaccharide can be selected. Molecularweights from 10,000 to 5,000,000 may be used. In one aspect, thepolysaccharide has a molecular weight of over 10,000. In another aspect,the polysaccharide has a molecular weight in the range of 10,000 to50,000. In another aspect, the polysaccharide has a molecular weight inthe range of 50,000 to 200,000. In another aspect, the polysaccharidehas a molecular weight in the range of 200,000 to 600,000. In an aspect,the polysaccharide has a molecular weight in the range of 600,000 to1,000,000. In an aspect, the polysaccharide has a molecular weight inthe range of 1,000,000 to 2,500,000. In yet another aspect, thepolysaccharide has a molecular weight in the range of 2,500,000 to5,000,000. The molecular weight can be measured by known methods,including, but not limited to, gel permeation chromatography orintrinsic viscosity.

After the functionalized polymer has been reacted with DVS to create afirst derivative of the polymer, this first derivative is then reactedwith a nucleophile, e.g., a thiol derivative, of a formula selected fromX—R¹ and X—R²—Y to provide a second derivative of the polymer. In theseformulae, R¹ is substituted or unsubstituted C₁-C₂₀ aliphatic oraromatic, R² is substituted or unsubstituted C₁-C₂₀ aliphatic oraromatic, X is a nucleophilic group, and Y is selected from carboxylicacid, sulfonic acid and hydroxyl. The nucleophile contains a thiol groupas X in a thiol derivative. In general, the thiol derivative can be asingle compound or a mixture of thiol compounds. Examples are alkylthiols, which may be, e.g., linear, branched, or cyclic, such asmethanethiol, ethanethiol, etc. Alternatively, the thiol may be an arylthiol, a charged thiol, a polymeric thiol, peptides with thiol groups,proteins with thiol groups, heterocycles with thiol groups, drugs, e.g.,active pharmaceutical ingredients, that contain thiol groups, growthfactors with thiol groups, and biologically active agents with thiolgroups.

For example, thiol compounds that can be used in the present disclosureare compounds that contain at least one free thiol group that is capableof reaction with a vinyl sulfone group via a Michael addition reaction.

The thiol compound may be identified by the formula R₁SH or R₂SH, whereR₁ and R₂ may be an aliphatic or aromatic moiety, either of which mayhave one or more substituents, e.g., be a substituted aliphatic moietyor a substituted aromatic moiety. An aliphatic moiety refers to an alkylor cycloalkyl moiety, either having 1-20 carbon atoms.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to the specified number of carbon atoms,and which is attached to the rest of the molecule by a single bond,e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl,n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, andthe like. In one aspect the alkyl group has 1 carbon. In one aspect thealkyl group has 2 carbons. In one aspect the alkyl group has 3 carbons.In one aspect the alkyl group has 4 carbons. In one aspect the alkylgroup has 4 carbons. In one aspect the alkyl group has 5 carbons. In oneaspect the alkyl group has 6 carbons. Two or more of these aspects maybe combined to describe derivatives of the disclosure.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,which may include fused or bridged ring systems, having from three tofifteen carbon atoms, preferably having from three to ten carbon atoms,and which is saturated or unsaturated and attached to the rest of themolecule by a single bond. Monocyclic radicals include, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Polycyclic radicals include, for example, adamantyl,norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.Unless otherwise stated specifically herein, a cycloalkyl group may beoptionally substituted by one or more substituents independentlyselected at each occurrence.

An aromatic moiety refers to a carbocyclic aromatic moiety, a.k.a., anaryl moiety, or a heteroaromatic moiety, a.k.a., a heteroaryl moiety,either having 1-20 carbon atoms, the heteroaromatic moiety having atleast one heteroatom selected from sulfur, oxygen and nitrogen.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. In one aspect thearyl ring system has 6 to 12 carbon atoms. In one aspect the aryl ringsystem has 6 to 10 carbon atoms. For purposes of this disclosure, thearyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclicring system, which may include fused or bridged ring systems. Arylradicals include, but are not limited to, aryl radicals derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, an aryl group may be optionally substituted by one ormore substituents independently selected at each occurrence.

“Heteroaryl” refers to “aryl” as defined herein, wherein the aromaticring includes one or more heteroatoms, preferably selected from N, O andS. Thus, a heteroaryl radical refers to an aromatic ring system radicalwherein the ring atoms are selected from carbon, nitrogen, oxygen andsulfur, and include at least one of nitrogen, oxygen and sulfur. Forpurposes of this disclosure, the heteroaryl radical may be a monocyclic,bicyclic, tricyclic or tetracyclic ring system, which may include fusedor bridged ring systems. Optionally, the heteroaryl radical is a 5-, 6-or 7-membered heteroaryl group. When there are multiple O and S atoms inthe heteroaryl ring system, the O atoms and/or S atoms are preferablynot linked directly to one another. Exemplary heteroaryl groups include5-membered rings, such as pyrrole, pyrazole, imidazole, 1,2,3-triazole,1,2,4-triazole, tetrazole, furan, thiophene, selenophene, oxazole,isoxazole, 1,2-thiazole, 1,3-thiazole, 1,2,3-oxadiazole,1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole. The heteroarylgroup may be a 6-membered ring, such as pyridine, pyridazine,pyrimidine, pyrazine, 1,3,5-triazine, 1,2,4-triazine, 1,2,3-triazine,1,2,4,5-tetrazine, 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, or fused ringsincluding a 6-membered ring such as indole, isoindole, indolizine,indazole, benzimidazole, benzotriazole, purine, naphthimidazole,phenanthrimidazole, pyridimidazole, pyrazinimidazole,quinoxalinimidazole, benzoxazole, naphthoxazole, anthroxazole,phenanthroxazole, isoxazole, benzothiazole, benzofuran, isobenzofuran,dibenzofuran, quinoline, isoquinoline, pteridine, benzo-5,6-quinoline,benzo-6,7-quinoline, benzo-7,8-quinoline, benzoisoquin-oline, acridine,phenothiazine, phenoxazine, benzopyridazine, benzopyrimi-dine,quinoxaline, phenazine, naphthyridine, azacarbazole, benzocarboline,phenanthridine, phenanthroline, thieno[2,3b]thiophene,thieno[3,2b]thiophene, dithienothiophene, isobenzothiophene,dibenzothiophene, and benzothiadiazo-thiophene. Unless stated otherwisespecifically in the specification, the ring atoms of a heteroaryl groupmay be optionally substituted by one or more substituents independentlyselected at each ring atom.

A substituted C₁-C₂₀ aliphatic or aromatic moiety refers to a C₁-C₂₀aliphatic or aromatic moiety having one or more substituents, where a“substituent” refers to monovalent group that may be attached to amentioned moiety. For example, a “substituted phenyl” refers to a phenylring having 1, 2, 3 or 4 substituents attached to the phenyl ring.Substituents may be selected from halogen, C₁-C₆alkyl, C₁-C₆haloalkyl,C₁-C₆hydroxyalkyl, —OH, —O(C₁-C₆alkyl), —O(C₁-C₆haloalkyl),—O(C₁-C₆hydroxyalkyl), —S(C₁-C₆alkyl), —S(C₁-C₆haloalkyl),—S(C₁-C₆hydroxyalkyl), cyano, amino (—NH₂), formyl (—CHO), carboxylicacid (—COOH), carboxylate ester (—COOR where R is a C₁-C₁₀ alkyl group).

These thiol compounds include alkyl thiols which may be linear, branchedor cyclic, aryl thiols, charged thiol compounds, polymers that contain afree thiol, peptides that contain a free thiol, heterocycles thatcontain a free thiol, drugs or biologically active compounds with a freethiol, growth factors with a free thiol, antibodies or antibodyfragments with a free thiol and proteins with a free thiol. Examples ofsuch thiol compounds include, and are not limited to thiophenol,2-phenylethanethiol, triphenylmethanethiol, 4-methylbenzenethiol,4-aminothiophenol, 2-aminothiophenol, 4-methoxy-α-toluenethiol,4-nitrothiophenol, 4-tert-butylbenzenethiol, 2-mercapto-2-phenylaceticacid, 4-mercaptobenzoic acid, 2-mercaptobenzoic acid (thiosalicylicacid), 3-mercapto-1-propanol, 1-mercapto-2-propanol,4-mercapto-1-butanol, 3-mercapto-1-hexanol, 6-mercapto-1-hexanol,8-mercapto-1-octanol, 9-mercapto-1-nonanol, 11-mercapto-1-undecanol,4-mercapto-4-methylpentan-2-ol, ethanethiol, 1-propanethiol,2-propanethiol, 1-butanetiol, 1-Pentanethiol, 1-hexanethiol,2-ethylhexanethiol, 1-heptanethiol, 1-octanethiol, 1-nonanethiol,1-decanethiol, 1-undecanethiol, 1-dodecanethiol, 1-tetradecanethiol,1-hexadecanethiol, cis-9-octadecene-1-thiol, 1-octadecanethiol,2-methyl-1-butanethiol, 3-methyl-1-butanethiol, cycloalkyl,cyclohexanethiol, cyclopentanethiol, sodium3-mercapto-1-propanesulfonate, sodium mercaptopyruvate,6-mercaptohexanoic acid, 8-mercaptooctanoic acid , 11-mercaptoundecanoicacid, 16-mercaptohexadecanoic acid, sodium 2-mercaptoethanesulfonate,3-mercaptopropionic acid, 2-amino-4-mercaptobutyric acid(DL-homocysteine), L-cysteine, 11-mercaptoundecylphosphoric acid,2-mercapto-1-methylimidazole, 1-benzyl-2-mercaptoimidazole,2-mercapto-6-methylpyridine, 3-mercapto-2-butanone,3-mercapto-3-methyl-1-butyl-1-formate, 3-mercapto-3-methylbutan-1-ol,7-mercapto-4-methylcoumarin, 2-mercapto-4-methyl-5-thiazoleacetic acid,2-mercapto-5-nitrobenzimidazole, 2-mercapto-5-benzimidazolesulfonic acidsodium salt dihydrate, 3-mercapto-N-nonylpropionamide,2-mercapto-4-methylpyrimidine hydrochloride, 2-mercapto-2-phenylaceticacid, 2-mercapto-3-(trifluoromethyl)pyridine,2-mercapto-N-m-tolylacetamide, and 4-mercapto-4-methylpentan-2-ol areexemplary thiol compounds.

Polymers with free thiols include but are not limited toThiol-PEG3-phosphonic acid, poly(L-lactide), thiol terminated 5000,poly(L-lactide), thiol terminated 2500, PEG-SH 3000, PEG-SH 5000,thiol-functionalized hyaluronic acid, thiol-functionalized chitosan,thiol functionalized alginate, thiol functionalized dextran, thiolfunctionalized chondroitin sulfate and thiol functionalizedcarboxymethyl cellulose.

Examples of thiol functionalized hyaluronic acid include but are notlimited to a thiol group linked to hyaluronic acid through a hydrazidecompound as described in U.S. Pat. No. 7,981,871, through carbodiimidegroups as described in U.S. Pat. No. 6,884,788, as well as thosedescribed in U.S. Pat. No. 8,124,757.

Examples of thiol functionalized chitosan include but are not limited tochitosan-cysteine conjugates, chitosan-thioglycolic acid conjugates andchitosan-4-thio-butylamidine conjugates.

Non-degradable thiol functionalized polymers include but are not limitedto polycarbophil-cysteamine conjugates, polycarbophil-cysteineconjugates, and poly(acrylic acid)-homocysteine conjugates.

Thiolated peptides or peptides that contain at least of free thiol,include but are not limited to a cysteine terminated peptide containingresidues 73-92 of the knuckle epitope of BMP-2 (N→C:KIPKASSVPTELSAISTLYLSGGC), thiolated gelatin (see, e.g., U.S. Pat. Nos.7,928,069 and 7,981,871), cysteine terminated cell adhesion epitopessuch as Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGDS) andIle-Lys-Val-Ala-Val (IKVAV), cysteine terminated TAT peptide(GRKKRRQRRRPQ), laminin peptide sequenceCys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg(CSRARKQAASIKVAVSADR; lam-IKVAV), and cysteine terminated Elastin-likepolypeptides such as those of the sequence (V P G X G)n where X=anyamino acid except proline.

Thiol containing drugs include but are not limited to Captopril,Thiorphan, Tiopronin and Penicillamine.

Suitable proteins that contain a cysteine group include but are notlimited to an IL-3 variant (see, e.g., U.S. Pat. No. 5,166,322), an IL-2variant (see, e.g., U.S. Pat. No. 5,206,344), protease nexin-1 varients(see, e.g., U.S. Pat. No. 5,766,897), Cysteine variants ofgranulocyte-macrophage colony-stimulating factor (see, e.g., U.S. Pat.No. 7,148,333; and Bioconjugate Chem., 2005, 16 (5), pp 1291-1298; DOI:10.1021/bc050172r), cysteine modified maize ribosome-inactivatingprotein (maize RIP) [see, e.g., Toxins 2016, 8, 298;doi:10.3390/toxins8100298], cysteine analog of erythropoietin [see,e.g., Int J Nanomedicine. 2011; 6: 1217-1227; doi: 10.2147/IJN.S19081],reduced antibody fragments [see, e.g., Protein Eng Des Sel (2007) 20(5): 227-234.DOI: https://doi.org/10.1093/protein/gzm015], and cysteineanalogues of Bone Morphogenetic Protein-2 (see, e.g., BioconjugateChem., 2010, 21 (10), pp 1762-1772; DOI: 10.1021/bc9005706.

Suitable growth factors that comprise a free thiol group include but arenot limited to Cysteine Analogs of Human Basic Fibroblast Growth Factor(hbFGF) [see, e.g., Tropical Journal of Pharmaceutical Research October2014; 13 (10): 1601-1607;

http://dx.doi.org/10.4314/tjpr.v13i10.5; and Protein Expr. Purif. 2006July; 48(1):24-7https://doi.org/10.1016/j.pep.2006.02.002]).

In one aspect, the present disclosure provides a process comprising:reacting hydroxyl groups attached to a polymer, such as hydroxyl groupson hyaluronic acid (HA), with divinyl sulfone (DVS) to provide a firstderivative of the polymer; and reacting the first derivative of thepolymer with a nucleophile of a formula selected from X—R¹ and X—R²—Y toprovide a second derivative of the polymer. The first derivative willhave a number of ethenyl (vinyl) groups attached to sulfone groups thatare, in turn attached through an oxyethylene group to the polymer. Someor all of these vinyl groups are reacted with a nucleophilic compound,e.g., a thiol derivative as described above. The extent to which thesevinyl groups undergo reaction may be specified according to the presentdisclosure. In one aspect, all or nearly all, e.g., 100%, or 99-100%, or98-100%, or 97-100%, or 96-100%, or 95-100% are substituted with thethiol derivative. In another aspect, partial substitution is achievedwith the thiol derivative, e.g., 1-95% of the free available vinylsulfone groups are derivatized.

For example, in one aspect the number of vinyl sulfone residues, thatare attached to the polysaccharide, and that can be reacted with a freethiol-containing compound can be altered. The percentage of the residualvinyl sulfone groups reacted with a free thiol-containing compound canvary from 1% to 100%. NMR, such as ¹H-NMR, can be used to determine thepercent substitution. When 100% substitution of the vinyl sulfone groupsoccurs, essentially all of the available vinyl sulfone residues attachedto the polysaccharide have reacted with the free thiol -containingcompound to form a thioether linkage. If less than 100% of the availablevinyl sulfone groups react with the free thiol-containing compound, thepolysaccharide will comprise both vinyl sulfone groups as well ascompounds attached via a thioether linkage. The fraction of the repeatunits of the polysaccharide that are substituted through a thioetherlinkage can be determined by NMR, usually ¹H-NMR. The percentsubstitution, often calculated on a molar basis, can range from 1% to100%, preferably greater than 10% and more preferably greater than 25%.

In one aspect, the Michael addition reaction of a free-thiol compoundwith the vinyl sulfone residue on the polysaccharide can occur using asingle free-thiol containing compound. In another aspect, the additionreaction can occur using more than 1 free thiol-containing compound inwhich the free thiol-containing compounds are different from each other.

FIG. 2 illustrates options for performing polymer derivatizationreactions according to the present disclosure. In FIG. 2, “A” identifiesa hydroxyl-substituted polymer such as hyaluronic acid orpolyvinylalcohol. The polymer A may be characterized in terms ofmolecular weight. In one aspect, the intrinsic viscosity of polymer A isused as an indicator of the polymer's molecular weight. Optionally, theintrinsic viscosity of polymer A is in the range of 0.3 to 3 m³/Kg. Inanother aspect, chromatography is used to characterize the molecularweight of polymer A. Optionally, the weight average molecular weight ofpolymer A is approximately 75,000 Da to 3,000,000 Da.

In FIG. 2, “B” identifies the product of reacting polymer A with divinylsulfone (DVS) under basic conditions (NaOH in an aqueous solvent).Polymer B is a compound of the present disclosure. Polymer B is shown astwo polymers. As joined together through X linkages, where X representsa diethyl sulfone group of the formula —CH₂—CH₂—SO₂—CH₂—CH₂— which islinked at each of its ends to an oxygen atom that was formerly part of ahydroxyl group from polymer A. The X groups are created by reaction oftwo hydroxyl groups reacting with two vinyl groups of divinyl sulfone.The X groups are shown linking together two different A polymers,however an X group may also link together two hydroxyl groups of asingle A polymer to provide a polymer B according to the presentdisclosure.

In FIG. 2, “B” contains three X linkages between two A polymers inaddition to three VS groups. A VS group is the result of a divinylsulfone substitution reaction with a hydroxyl group of an A polymer. Inorder to create a VS group, one and only one of the two vinyl groups ofa divinyl sulfone molecule reacts with one and only one hydroxyl groupof a polymer A. In one aspect of the present disclosure,hydroxy-substituted polymers (“A”) are reacted with divinyl sulfone(DVS) to create linkages between two or more hydroxyl groups in amixture of hydroxyl substituted polymer chains, and additionally tocreate vinyl sulfone substituents on one or more hydroxyl-substitutedpolymer chains (shown as polymer B in FIG. 2).

In one aspect, the polymer B still contains unreacted hydroxyl groups.For example, when a flask is charged with a desired amount of polymer Acomprising a specified number of hydroxyl groups, the addition of DVSwill consume at least 5%, or least 10%, or at least 20%, or at least30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%,or at least 80% of those initial hydroxyl groups in the formation of Xand VS groups present in polymer B. The number of hydroxyl groupspresent after reaction of DVS may also, or alternatively be described interms of the residual hydroxyl groups, so that at least 15%, or at least20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%,or at least 50%, or at least 60%, or at least 70%, or at least 80% ofthe initial hydroxyl groups are still present in polymer B. The numberof hydroxyl groups present in polymer B may also be expressed as a rangeof the initial number of hydroxyl group present in polymer A, e.g., theconversion of polymer A to polymer B may consume 5-10% of the availablehydroxyl groups, or in other aspects, 5-15%, or 5-20%, or 5-25%, or5-30%, or 5-35%, or 10-15%, or 10-20%, or 10-25%, or 10-30%, or 10-35%,or 10-40% of the initially available hydroxyl groups.

In one aspect, the polymer B contains both X and VS substituents. In oneaspect, the polymer B contains both X and VS substituents in a molarratio of where the number of VS groups exceeds the number of X groups.However, in another aspect, the number of X groups exceeds the number ofVS groups. In other aspects, the X groups provide at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the total number of Xand VS groups.

As shown in FIG. 2, polymer B may serve as a reactant to create eitherpolymer C or polymer D, each of which is a polymer according to thepresent disclosure. To create polymer C, a mixture of nucleophiliccompounds, represented as R₁SH and R₂SH in FIG. 2, is reacted withpolymer B. To create polymer D, a single nucleophilic compound,represented as R₁SH in FIG. 2, is reacted with polymer B. The presentdisclosure provides polymer B, polymer C, polymer D as well as reactionsto create polymer B from polymer A, reactions to create polymer C frompolymer B, and reactions to create polymer D from polymer B. In oneaspect, each of polymers A, B and C is a derivatized hyaluronic acid.

Polymer D contains X moieties which link together two polymer A chains.In addition, polymer D contains Z—S—R₁ moieties which are created by thereaction of the vinyl sulfone (VS) groups of polymer B with thiolcompound R₁SH to provide —O—CH₂—CH₂—SO₂—CH₂—CH₂—S—R₁ moieties, which areabbreviated as Z—S—R1 moieties in FIG. 2. In one aspect, the presentdisclosure provides polymer D having a mixture of X groups and Z—S—R₁groups. In one aspect, X groups provide at least 10%, or at least 20%,or at least 30%, or at least 40%, or at least 50%, or at least 60%, orat least 70%, or at least 80%, or at least 90% of the total of the X andZ—S—R₁ groups. In one aspect, Z—S—R₁ groups provide at least 10%, or atleast 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90% of thetotal of the X and Z—S—R₁ groups.

In one aspect, the present disclosure provides polymer E having astructure as set forth in FIG. 2. In one aspect, the present disclosureprovides polymer F having a structure as identified in FIG. 2. Inanother aspect, the present disclosure provides polymer G having astructure as identified in FIG. 2. In yet another aspect, the presentdisclosure provides polymer H having a structure as identified in FIG.2.

As shown in FIG. 2, polymer A may be reacted with divinyl sulfone underbasic conditions to provide polymer E. As shown in FIG. 2, polymer E maybe formed from polymer A by reaction of the hydroxyl groups of polymer Awith divinyl sulfone (DVS) to convert them to vinyl sulfone (VS) groups.In polymer E, there are few, if any, X groups which link together twohydroxyl groups of polymer A. In various aspects, the VS groupsconstitute at least 80%, or at least 85%, or at least 90%, or at least,95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%,or at least 99.5%, or at least 99.9% of the total of X and VS groupspresent in polymer E.

Polymer E may be reacted with R₁SH, optionally in combination with oneor more additional nucleophilic compounds, e.g., R₂SH, to providepolymers of structure F, G or H, as shown in FIG. 2. Polymer F has amixture of residual VS groups and Z—S—R1 groups formed by reaction of VSgroups with R₁SH. The charge of R₁SH is less than 100% of the totalnumber of VS groups present on polymer E, calculated on a molar basis.Based on this stoichiometry, not all of the VS groups will react withR₁SH molecules, and accordingly polymer F has a mixture of VS and Z—S—R₁groups. In one aspect, VS groups provide at least 10%, or at least 20%,or at least 30%, or at least 40%, or at least 50%, or at least 60%, orat least 70%, or at least 80%, or at least 90% of the total of the VSand Z—S—R1 groups. In one aspect, Z—S—R₁ groups provide at least 10%, orat least 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90% of thetotal of the VS and Z—S—R1 groups.

Polymer G has a majority of Z—S—R₁ groups, and little or no X and VSgroups. In various aspects, the Z—S—R₁ groups constitute at least 80%,or at least 85%, or at least 90%, or at least, 95%, or at least 96%, orat least 97%, or at least 98%, or at least 99%, or at least 99.5%, or atleast 99.9% of the total of X, VS and Z—S—R₁ groups present in polymerG. Polymer G may be formed by reaction of polymer E and an equimolar ormolar excess of R₁SH molecules, based on the moles of available VSgroups.

Polymer H has a majority of Z—S—R₁ and Z—S—R₂ groups, and little or no Xand VS groups. In various aspects, the total of the Z—S—R₁ and Z—S—R₂groups constitute at least 80%, or at least 85%, or at least 90%, or atleast, 95%, or at least 96%, or at least 97%, or at least 98%, or atleast 99%, or at least 99.5%, or at least 99.9% of the total of X, VS,Z—S—R1 and Z—S—R₂ groups present in polymer H. Polymer H may be formedby reaction of polymer E and a mixture of nucleophilic compounds, e.g.,a mixture of R1SH and R₂SH, such as shown in FIG. 2.

In one aspect, the present disclosure provides polymer I having astructure as set forth in FIG. 2. In one aspect, the present disclosureprovides polymer J which is a gel prepared as shown in FIG. 2. Inanother aspect, the present disclosure provides polymer K which is a gelprepared as shown in FIG. 2.

Polymer I has a mixture of Z—S—R₁ and VS substituents. In one aspect,the present disclosure provides polymer I having a mixture of VS groupsand Z—S—R₁ groups. In one aspect, VS groups provide at least 10%, or atleast 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90% of thetotal of the VS and Z—S—R₁ groups. In one aspect, Z—S—R₁ groups provideat least 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90% of the total of the VS and Z—S—R₁ groups. Polymer I may beformed by reacting polymer G with divinylsulfone under basic conditions.This reaction converts hydroxyl groups present on polymer G (not shownin FIG. 2) to vinyl sulfone (VS) groups.

Polymers L and K are gels which may be prepared as shown in FIG. 2.Polymer J may be formed by crosslinking polymer G. Polymer K may beformed by crosslinking polymer H.

In one aspect, the present disclosure provides polymer L having astructure as set forth in FIG. 2. In another aspect, the presentdisclosure provides polymer M having a structure as identified in FIG.2. In yet another aspect, the present disclosure provides polymer Nhaving a structure as identified in FIG. 2.

Polymer Las shown in FIG. 2 contains a mixture of Z—S—R₁ and Z—S—R₂substituents. Polymer L may additionally contain hydroxyl substituents(not shown). In various aspects, the total of the Z—S—R₁ and Z—S—R₂groups constitute at least 80%, or at least 85%, or at least 90%, or atleast, 95%, or at least 96%, or at least 97%, or at least 98%, or atleast 99%, or at least 99.5%, or at least 99.9% of the total of X, VS,Z—S—R₁ and Z—S—R₂ groups present in polymer L. Polymer L may be formedby reaction of polymer F, which contains Z—S—R₁ and VS substituents,with R₂SH, to thereby convert the VS substituents to Z—S—R₂substituents.

Polymer M as shown in FIG. 2 contains a mixture of X, Z—S—R1 and Z—S—R₂groups. Polymer M may additionally contain hydroxyl substituents (notshown). Polymer M may be formed by adding a crosslinker, such asdivinylsulfone, to Polymer L that contains residual hydroxyl groups. Thecrosslinker creates X groups between hydroxyl groups present on polymerL.

Polymer N as shown in FIG. 2 contains a mixture of Z—S—R₁ and —R—groups, where an R group forms a linkage between different polymer Achains. The R groups may be introduced by reacting a precursor polymer,such as polymer F or other polymer containing VS groups, with apolyfunctional nucleophile, such as R(SH)n where n is greater than orequal to 2. In R(SH)n, R represents an aliphatic or aromatic group thatis optionally substituted.

In one aspect, the present disclosure provides polymer O which is a gelthat may be formed as shown in FIG. 2. In another aspect, the presentdisclosure provides polymer P which is a gel that may be formed as shownin FIG. 2.

Polymer O may be formed from Polymer I by a two-step reaction. In afirst step, polymer I is reacted with a nucleophilic compound, such asR₁SH, to convert VS groups present on polymer I, into the correspondingZ—S—R₁ groups. In a second step, a crosslinker X is added to thisintermediate polymer to provide a polymeric gel O.

Polymer P may be formed from Polymer I by a two-step reaction. In afirst step, polymer I is reacted with a nucleophilic compound, such asR₂SH, to convert VS groups present on polymer I, into the correspondingZ—S—R₂ groups. In a second step, a crosslinker X is added to thisintermediate polymer to provide a polymeric gel P.

Polymer I may also serve as a precursor to a crosslinked polymer having—R— groups as the linkage between polymer chains. The R groups may beintroduced by reacting a polymer I, or another polymer containing VSgroups, with a polyfunctional nucleophile, such as R(SH)n where n isgreater than or equal to 2. In R(SH)n, R represents an aliphatic oraromatic group that is optionally substituted.

FIG. 3 illustrates options for performing polymer derivatizationreactions according to the present disclosure. In FIG. 3, “A” identifiesa hydroxyl-substituted polymer such as hyaluronic acid orpolyvinylalcohol, which is likewise shown as polymer A in FIG. 2.However, in contrast to FIG. 2, the reaction schemes of FIG. 3 begin byperforming a crosslinking reaction on polymer A, and achieving little orno conversion of hydroxyl groups on polymer A into an alternativemonofunctional reactive group.

As shown in FIG. 3, polymer A may be reacted with a crosslinking agent,to provide a crosslinked version of polymer A, which is denoted aspolymer B in FIG. 3. Suitable crosslinking reactions forhydroxyl-containing polymers are described elsewhere herein.

In one aspect, the present disclosure provides polymer C having astructure as set forth in FIG. 3. In another aspect, the presentdisclosure provides polymer D having a structure as identified in FIG.3. In yet another aspect, the present disclosure provides polymer Ehaving a structure as identified in FIG. 3.

Polymer C may be formed by reacting polymer B with divinyl sulfone (DVS)under basic conditions. Under these reaction conditions, hydroxyl groupspresent on polymer B (not shown) react with DVS to convert hydroxylgroups to VS groups. In one aspect, VS groups provide at least 10%, orat least 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90% of thetotal of the VS and X groups present in polymer C. In one aspect, Xgroups provide at least 10%, or at least 20%, or at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or at least 90% of the total of the VS and X groups presentin polymer C.

Polymer D in FIG. 3 is a crosslinked polymer having both Z—S—R₁ andZ—S—R₂ substituents. Polymer D may be formed by reacting polymer C witha mixture of nucleophilic compounds, such as R₁SH and R₂SH as shown inFIG. 3. In one aspect, the total of the Z—S—R₁ and Z—S—R₂ groups provideat least 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90% of the total of the Z—S—R₁, Z—S—R₂ and X groups present inpolymer D. In one aspect, X groups provide at least 10%, or at least20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%,or at least 70%, or at least 80%, or at least 90% of the total of theZ—S—R₁, Z—S—R₂ and X groups present in polymer D.

Polymer E in FIG. 3 is a crosslinked polymer having Z—S—R₁ substituents(but not having any and Z—S—R₂ substituents). Polymer E may be formed byreacting polymer C of FIG. 3 with a nucleophilic compound, such as R₁SHas shown in FIG. 3. In one aspect, the Z—S—R₁ groups provide at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90% ofthe total of the Z—S—R₁ and X groups present in polymer E. In oneaspect, X groups provide at least 10%, or at least 20%, or at least 30%,or at least 40%, or at least 50%, or at least 60%, or at least 70%, orat least 80%, or at least 90% of the total of the Z—S—R₁ and X groupspresent in polymer E.

As also shown in FIG. 3, the present disclosure provides polymers ofstructure F and of structure G, as well as crosslinked gels thereof.Polymer F of FIG. 3 contains Z—S—R1 substituents, while polymer Gcontains a mixture of Z—S—R1 and Z—S—R₂ substituents. Neither ofpolymers F or G are crosslinked polymers. However, each of polymers Fand G may be treated with a crosslinking agent, or exposing tocrosslinking conditions, to provide the corresponding crosslinkedpolymer which will have the form of a gel (identified as polymer H inFIG. 3).

Though not wishing to be bound by any particular theory, it is believedthat there may be none to a low level of crosslinking occurring whenvinyl groups are present in in a solution of derivatized polyhydricpolymer molecules. This “accidental” crosslinking may be present but isnot measurable and the solution does not exhibit characteristics ofcross-linking, and would be referred to herein as a solution orcomposition comprising non-cross-linked derivatives of polymericpolyhydric alcohols.

Thus, in one aspect, the present disclosure provides vinyl sulfonefunctionalization (i.e., derivatization) of a polysaccharide (“a firstderivative”) followed by reaction of the vinyl sulfone substituent withone or more free thiol-containing compounds (“a second derivative”)which is in turn followed by a second functionalization (i.e.,derivatization) reaction with divinyl sulfone to produce apolysaccharide that is functionalized with compounds through a thioetherlinkage as well as with vinyl sulfone functional groups (“a thirdderivative”). In another aspect, the above compounds can be furtherreacted with free thiol-containing compounds (“a fourth derivative”).The molar ratio of the free thiol-compound used for the reaction can bealtered such that 1% to 100% of the second added vinyl sulfonefunctional groups are reacted. The free thiol-containing compound thatis used in the second Michael addition reaction (derivatizationreaction) can be the same or it can be different from that used in thefirst Michael addition reaction. For the second Michael additionreaction, a single free thiol-containing compound can be used or amixture of 2 or more different free-thiol containing compounds can beused. In another aspect, at least one additional round of vinylsulfone/free thiol-containing compound reactions cycles can be performedusing the same free-thiol containing compound or one or more differentfree-thiol containing compounds

In one aspect, a process of the present disclosure further comprisescrosslinking a second derivative of the polymer, e.g., crosslinking byreacting the second derivative of the polymer with a crosslinking agent.Upon crosslinking, the second derivative is converted to a thirdderivative of the polymer, where the third derivative is a crosslinkedpolymer.

For example, in one aspect, a polysaccharide derivatized with one ormore free-thiol containing compounds and also comprises residualavailable vinyl sulfone functional groups can undergo crosslinking bysubjecting a solution of the derivatized polyhydric polymer to basicconditions that are sufficient to allow the residual available vinylsulfone group to react with a hydroxyl group of the polysaccharide. Inone aspect, the reaction pH is greater than 12 for example, in the 12.5to 13.0 pH range. The amount of residual vinyl sulfone functionalgroups, often measured as percent substitution as measured by ¹H-NMR,reaction time and reaction temperature can be selected to achieve thedesired degree of crosslinking.

In another aspect, a polysaccharide derivatized with one or morefree-thiol containing compounds and also comprises residual availablevinyl sulfone functional groups can be mixed with a polysaccharidederivatized with one or more free-thiol containing compounds and alsocomprises residual available vinyl sulfone functional groups wherein thefree-thiol containing compounds can be the same or different or acombination thereof. The resultant mixture can undergo crosslinking bysubjecting a solution of the derivatized polyhydric polymer to basicconditions that are sufficient to allow the residual available vinylsulfone group to react with a hydroxyl group of the polysaccharide. Inone aspect, the reaction pH is greater than 12, for example, in the 12.2to 13 pH range. The amount of residual vinyl sulfone functional groups,often measured as percent substitution as measured by ¹H-NMR, reactiontime and reaction temperature can be selected to achieve the desireddegree of crosslinking.

In another aspect, a non-derivatized polysaccharide can be added to thecrosslinking reaction mixtures described above and the resultant mixturecan undergo crosslinking by subjecting the solution of derivatizedpolymeric polyhydric alcohol and the non-derivatized polysaccharide tobasic conditions that are sufficient to allow the residual availablevinyl sulfone groups of the derivatized polymeric polyhydric alcohol toreact with a hydroxyl group of the polysaccharide. In one aspect, thereaction pH is greater than 12, for example, in the 12.2 to 13 pH range.The amount of residual vinyl sulfone functional groups, often measuredas percent substitution as measured by 1H-NMR, reaction time andreaction temperature can be selected to achieve the desired degree ofcrosslinking.

Crosslinking may be achieved by using an external crosslinking agent. Inone aspect, a crosslinking agent is added to the second derivative ofthe polymer. Exemplary crosslinking agents that could be used include:carbodiimides, bisepoxides, divinyl sulfone derivatives, andcombinations thereof. Another suitable crosslinking agent is a multiplethioether derivative. In one aspect, at least 2 (could be 2, 3, 4, etc.)different thioether derivatives are combined with a crosslinking agentand conditions are adjusted such that the derivatives of polyhydricpolymers become either fully crosslinked or partially crosslinked. Inthis case, exemplary crosslinking agents include, without limitation,carbodiimides, bisepoxides, divinyl sulfone derivatives and combinationthereof.

For example, in one aspect, a polysaccharide derivatized with one ormore free-thiol containing compounds can be crosslinked by adding acrosslinking agent and adjusting the pH of the reaction mixture suchthat the derivatized polysaccharide forms a crosslinked derivatizedpolysaccharide orcomposition. Crosslinking agents that can be usedinclude but are not limited to biscarbodiimides, bisepoxides, divinylsulfone derivatives, di-isocyanates, dihalide chlorides, disuccinimidylderivatives and combinations thereof.

Biscarbodiimide compounds can include but are not limited topara-phenylenebis-(ethyl)-carbodiimide, 1,6-hexamethylenebis(ethylcarbodiimide), 1,8-octamethylene bis(ethylcarbodiimide), 1,10decamethylene bis(ethylcarbodiimide), 1,12 dodecamethylenebis(ethylcarbodiimide), PEG-bis(propyl(ethylcarbodiimide)),2,2′-dithioethyl bis(ethylcarbodiimde), 1,1′-dithio-p-phenylenebis(ethylcarbodiimide); para-phenylene-bis(ethylcarbodiimide), and1,1′-dithio-m-phenylene bis(ethylcarbodiimide).

When utilizing a biscarbodiimide crosslinker, the biscarbodiimide ismixed with a buffered aqueous solution of the derivatized carboxylicacid containing polysaccharide. The target pH of the buffered solutioncan be between pH 5 and pH 6.5.

Bisepoxide compounds can include but are not limited to 1,4-butanedioldiglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane (DEO), poly(ethyleneglycol) diepoxide. When utilizing a bisepoxide crosslinker, thebisepoxide is mixed with an aqueous solution of the derivatizedpolysaccharide and the pH is raised to a pH>9. The reaction can becarried out at 40° C. for greater than 4 hours to produce a crosslinkedderivatized polyhydric polymer.

Divinyl sulfone crosslinking agents can include but are not limited todivinyl sulfone and poly(ethylene glycol) bisvinyl sulfone.

When utilizing a divinyl sulfone crosslinker, the reaction pH in anaqueous solution can be raised to a pH greater than 12 to effectcrosslinking. The degree of crosslinking can be altered by changing theamount of crosslinking agent added, reaction time, the reaction pH andreaction temperature.

In another aspect, a mixture of at least 2 different thioetherderivatized polysaccharides can be mixed together, a crosslinking agentcan be added and the reactions conditions adjusted such that thederivatized polyhydric polymers are crosslinked. The relative ratios ofthe different derivatized polysaccharides can be altered such thatcrosslinked derivatized polyhydric polymers with different propertiesare obtained. These properties include but are not limited toequilibrium swelling, swelling rate, drug release characteristics,elastic modulus, storage modulus, loss modulus, degradation, tensilestrength, tissue adhesiveness and lubricity. As used herein “derivatizedpolyhydric polymers” may also include compositions comprising one ormore derivatized polyhydric polymers.

In another aspect, at least 2 different crosslinking agents can be usedto crosslink the derivatized polysaccharide. In one aspect, twodifferent crosslinking agents from the same group could be used tocrosslink the derivatized polyhydric polymers. For example, divinylsulfone and poly(ethylene glycol) bisvinyl sulfone or 1,4-butanedioldiglycidyl ether (BDDE) and poly(ethylene glycol) diepoxide could beused.

In another aspect, two different crosslinking agents from differentgroups could be used. For example, divinyl sulfone and 1,4-butanedioldiglycidyl ether (BDDE) may be used to crosslink the derivatizedpolysaccharides. In another aspect, the crosslinker can be addedsequentially such that initial crosslinking occurs in the presence ofthe first crosslinked and then the second crosslinker is added such thatsecondary crosslinking occurs. The reaction conditions may be changedafter the first crosslinking reaction and prior to the secondcrosslinking reaction. Reaction conditions such as temperature, pH,buffer, ionic strength and solvent composition can be altered.

In one aspect, crosslinked derivatized polyhydric polymers can beprepared though ionic crosslinking. This can be accomplished by mixing aderivatized polyhydric polymer of this disclosure that has a negativecharge with a compound that has two or more positive charges. In oneaspect, a solution of the derivatized polyhydric polymer of thisdisclosure that has a negative charge can be prepared and then mixedwith a solution of a compound that has two or more positive charges.Inorganic compounds that can be used include but are not limited toferric chloride, aluminum chloride, chromium sulfate, and aluminumsulfate. Positively charged polymers that can be used include polymersthat comprise more than two lysines, arginine or histadine amino acids,chitosan and chitosan derivatives, deacetylated hyaluronic acid,polyethyleneimine (PEI), poly(N,N-dimethylaminoethylmethacrylate),poly(4-vinylpyridine), polyethyleneglycol-polylysine block copolymers(PEG-PLL), dextran grafted polylysine copolymers, or combinationsthereof.

In one aspect, the positively charged or the negatively charged polymercan first be applied. This can then be followed by application of theoppositely charged polymer such that at the interface of the two layers,ionic interactions occur such that the polymers are crosslinkedtogether. In another aspect, the process can be repeated at least onemore time.

A second derivative of the polymer (e.g.,HA-(OCH₂CH₂SO₂CH₂CH₂—X—R₁—Y)_(n)) may be crosslinked via internal andexternal crosslinking. For example, in one aspect, a polysaccharidederivatized with one or more free-thiol containing compound and alsocomprising residual available vinyl sulfone functional groups can becrosslinked in the presence of an external crosslinking agent. In oneaspect, the reaction conditions can be adjusted such that the residualavailable vinyl sulfone groups and the added external crosslinked reactsimultaneously. For example, divinyl sulfone can be added as theexternal crosslinker and then the pH can be increased to a pH>12 whichwill result is crosslinking.

As another example, the crosslinking via the residual available vinylsulfone functional groups can take place first which is then followed bythe addition of the external crosslinker. The reaction conditions, forexample pH, can be changed to effect the crosslinking reaction of theexternal added crosslinker. For example, the pH of the derivatizedpolysaccharide that contains the residual available vinyl sulfonefunctional groups can be raised to a pH>12. Once the reaction has beenreached the desired level, the pH can be changed to between pH 5 and pH6.5 with a buffer and then biscarbodiimide crosslinker, for examplepara-phenylenebis-(ethyl)-carbodiimide, can be added to the reactionmixture and allowed to react until the desired level of crosslinking isobtained. In another aspect, the biscarbodimide crosslinking can takeplace first by adjusting the pH of the derivatized polysaccharide tobetween 5 and 6.5, adding the biscarbodiimide, allowing the crosslinkingto proceed to the desired level, then raising the pH to pH>12 to allowthe residual vinyl sulfone functional groups to crosslink.

In one aspect, a polysaccharide derivatized with one or more free-thiolcontaining compound and also comprises residual available vinyl sulfonefunctional groups can be crosslinked in the presence of an externalcrosslinking agent that has at least two free thiol functional groups.These free thiol groups may be positioned upon a central molecule, “C”.The central molecule may be a linear or cyclic alkane, a polyethyleneglycol (PEG) oliogomer or polymer, or any other such suitable centralmolecule. In the case of PEG-based crosslinking agents, the PEG may belinear, branched (having two polymer arms), or multi-armed (e.g., having3, 4, 5, 6, 7, 8 or more polymer arms). Thus, in such instances, thecentral molecule will typically a linear PEG, a branched PEG having 2arms, or a multi-armed PEG having PEG arms emanating from a centralcore.

Illustrative cores for such multi-armed polymers include erythritol,pentaerythritol, trimethylolpropane, glycerol, glycerol dimer(3,3′-oxydipropane-1,2-diol), glycerol oligomers, sorbitol,hexaglycerol, and the like.

Illustrative thiol crosslinking agents include PEG-dithiol (HS-PEG-SH),3-arm PEG-tri-thiol (glycerine core), 4-arm PEG-tetrathiol(pentaerythritol core), or 8-arm PEG-octa-thiol (hexaglycerine core).The foregoing multi-armed PEG reagents may also have fewer than all armsfunctionalized with thiol. Additional suitable thiol reagents having PEGas the central molecule are available from Laysan Bio (Arab, Ala.), aswell as aromatic dithiols such as those available from NanoScience.Other suitable thiol crosslinking agents include dimercaptosuccinicacid, 2,3-dimercapto-1-propanesulfonic acid, Trimethylolpropanetris(3-mercaptopropionate), dithiol functionalized pluronics F127,dithiol functionalized F68, dihydrolipoic acid, peptides containing atleast 2 cysteine amino acids, thiol functionalized dextran, andthiol-functionalized hyaluronic acid.

The polymers of the present disclosure, e.g., the first, second andthird derivatives of a polymer such a polysaccharide, may be processedinto numerous forms. For the non-crosslinked derivatized polyhydricpolymers, exemplary compositions of the derivatized polyhydric polymersinclude, but are not limited to, a solution, a suspension, an emulsion,a film, a gel, a coating on a surface of an article, an electrospunmatrix, a microparticle, a fiber, a lyophilized solid, a rod, a disc, agel, a powder or in a particulate form. A particulate form can beprepared by milling (e.g., jet milling, roller milling, cryomilling,mechanical milling) fragmentation, spray drying, precipitation orgrinding. For the crosslinked derivatized polyhydric polymers,compositions of the derivatized polyhydric polymer can be as asuspension, a film, an electrospun matrix, a fiber, a lyophilized solid,a rod, a disc, a gel, a powder or in a particulate form. The particulateform can be prepared by milling (jet milling, roller milling,cryomilling, mechanical milling) fragmentation, spray drying,precipitation or grinding.

A solution of the derivatized polyhydric polymer can be prepared bydissolving the derivatized polyhydric polymer in an appropriate solventor a combination of solvents. For example, water or a combination ofwater and water-miscible solvent can be used. Water-miscible solventscan include but are not limited to methanol, ethanol, isopropanol,dimethyl formamide (DMF) acetone, 1,4-dioxane, pyridine, dimethylsulfoxide (DMSO), tetrahydrofuran (THF) and acetonitrile. The preparedsolutions can be sterilized by filtering through a 0.2 μm sterilefilter. In one aspect, a solution can be prepared using one derivatizedpolysaccharide. The concentration of the prepared solutions can rangefrom, e.g., 0.01% (w/v) to about 50% (w/v). In one aspect, theconcentration is in the 0.1% (w/v) to 10% (w/v) range.

A film of non-crosslinked derivatized polyhydric polymers of thisdisclosure can be prepared by preparing a solution of the derivatizedpolyhydric polymer. This solution can be then placed in a mold or drawnout on a surface using a gardner knife. The surface used can be glass,metal foil, stainless steel, Teflon, nylon, polyethylene, polypropyleneor a release liner. The solvent can then be removed to form the film.The rate of solvent removal can be altered by using at least one of thefollowing parameters: temperature, air or inert gas flow and pressure.To increase the rate of solvent evaporation, the temperature could beincreased, the air or inert gas flow rate could be increased or thepressure could be decreased. A combination of these process could alsobe used. To slow the rate of solvent evaporation, the temperature couldbe decreased, the air or inert gas flow rate could be reduced or thepressure could be increased. A combination of these process could alsobe used. A film can comprise one of the derivatized polyhydric polymersof this disclosure. The films can also comprise two or more differentderivatized polyhydric polymers of this disclosure. A composite film canbe prepared by preparing a first film and then casting a second film ontop of the first film. A composite film can be prepared by castingadditional layers sequentially on top of the previous layer. The layersof the composite film can comprise the same derivatized polyhydricpolymer if the disclosure, different derivatized polyhydric polymers ofthis disclosure or a combination thereof.

Lyophilized forms of the non-crosslinked derivatized polyhydric polymersof this disclosure can be prepared by making a solution of thederivatized polyhydric polymer, freezing the solution and then placingthe frozen derivatized polyhydric polymer solution under a vacuum suchthat the solvent is sublimed off to leave the derivatized polyhydricpolymer composition in the solid form. A lyophilized form of thederivatized polyhydric polymer composition of this disclosure cancomprise one of the derivatized polyhydric polymers of this disclosure.In another aspect, the lyophilized form of the derivatized polyhydricpolymers and/or compositions of this disclosure can comprise two or moredifferent derivatized polyhydric polymers of this disclosure. In anotheraspect, a lyophilized composition of one or more derivatized polyhydricpolymers can comprise one or more derivatized polyhydric polymers inaddition to an additive such as chitosan or chitosan derivatives (e.g.chitosan HCL, chitosan acetate, or chitosan lactate). The form of thelyophilized derivatized polyhydric polymer compositions may be dependenton the form of the container into which the solution was poured. Inanother aspect, the form of the lyophilized derivatized polyhydricpolymer may be dependent on the form of the contained into which thesolution was poured and frozen. The form can be a rectangle, square,disk, triangle, trapezoid, rod or any other form in which a mold can bemade.

A derivatized polyhydric polymer compositions of this disclosure can bein the form of a powder or particulate. The powder or particulate may beobtained directly via precipitation. A powder or particulate form canalso be obtained through a milling, grinding, spray drying orfragmentation process. Compositions, including but not limited to,films, precipitated derivatized polyhydric polymers, dried derivatizedpolyhydric polymers and/or compositions, lyophilized derivatizedpolyhydric polymers and/or compositions, or derivatized polyhydricpolymers and/or compositions, dried in a form can be further process viaa milling process (jet milling, roller milling, cryomilling, mechanicalmilling), a grinding or a fragmentation process. A combination of theseprocesses can be used. Derivatized polyhydric polymer composition withparticle size in the range of 100 nm to 5 mm can be prepared. Specificsize ranges of the powdered or particulate derivatized polyhydricpolymer compositions of this disclosure can be prepared by separatingthe derivatized polyhydric polymer composition particles according tosize using sieves. The distribution of particle sizes can be broad witha standard deviation of the average size of greater than 40%. Thedistribution of particle sizes can be narrow with a standard deviationof the average size of less than 30%. The final powdered or particulateform of the derivatized polyhydric polymer compositions of thisdisclosure can comprise a single distribution of average particle sizesor it can comprise two or more distributions of particles prepared bymixing particles of different average particle size.

A derivatized polyhydric polymer compositions of this disclosure can beformed into a solid form by preparing a solution of a derivatizedpolyhydric polymer in a solvent that can be removed, pouring thissolution into a mold of a specific shape and then removing the solventsuch that a solid form of derivatized polyhydric polymer composition isobtained. The molds used can be of various shapes and can include butare not limited to cubes, rectangles, rods, semi-circular rode andtubes. The solid derivatized polyhydric polymer composition of thisdisclosure can then be removed from the mold.

A derivatized polyhydric polymer composition of the disclosure can beprocessed into an electrospun matrix. In this process, a solution of thederivatized polyhydric polymer of the disclosure is prepared. Thesolvent used can be an organic solvent, water or a combination thereof.For example, for hyaluronic acid based derivatized polyhydric polymers,water/ethanol or water/dimethylformamide (DMF) solvent mixtures can beused. Solutions with a concentration of 0.5 to 5% (w/v) can be prepared.The solution that is to be electrospun can be placed in a syringe with aneedle. The syringe is then placed in a syringe pump. The needle canhave a blunt end and an inner diameter in the range of 0.25 to 1 mm. Theneedle and collection plate are attached to a high voltage supply. Avoltage is then applied to the system. The applied voltage can be in the10 kV to 45 kV. The syringe pump can extrude the solution. The flow rateof the syringe pump can be in the range of 10 uL/min to 1000 uL/min. Thecollector plate can be static, rotating or moving in a specific lineardirection to give the fibers some directional orientation. The shape ofthe collector plate can be varied with the collector plate having butnot limited to the following shapes: a flat surface, a textured surface,a curved surface, a square rod, a rectangular rod, a round mandrel, anoval mandrel, a semi-circular mandrel or a combination of these shapes.The temperature of the solution can be controlled as well as thecollection plate and the surrounding environment. The distance of theneedle tip to the collector plate can be altered. The distance of theneedle tip to the collector plate can be in the 2-20 cm range. Thecollection plate can also be submerged in or sprayed with a solvent thatassists in the precipitation of the newly spun fibers. For example, anethanol bath may be used during the electrospinning of hyaluronic acidderivatized polyhydric polymers and/or compositions of this disclosure.

A derivatized polyhydric polymer of the disclosure can be incorporatedthrough a solution coating or submersion of an electrospun matrixproduced in the following manner. In this process, single or multiplepolymer solutions are prepared. The polymers used can be biodegradablepolymers then include but are not limited to polyester, polyanhydride,polyorthoester, polycarbonate, poly-ester-co-carbonate),polyhydroxybutyrates or combinations thereof. Biodegradable polymers caninclude polylactice-co-glycolide copolymers, polydioxanone,polylactide-trimethylene carbonate copolymers as well as copolymers thatcomprise repeat units derived from at least one of the followingmonomers: l-lactide, dl-lactide, glycolide, trimethylene carbonate,epsilon-caprolactone, p-dioxanone and a morpholinedione.

The solvents used can be an organic solvent, water or a combinationthereof. For example, HFIP, DMSO, NMP, Chloroform, acetic acid, ethanol,dimethylformamide (DMF) solvents or mixtures of solvents can be used.Solutions with a concentration of 0.5 to 25% (w/v) can be prepared. Thesolution that is to be electrospun can be placed in a syringe with aneedle. The syringe is then placed in a syringe pump. The needle canhave a blunt end and an inner diameter in the range of 0.25 to 2.5 mm.The needle and collection plate are attached to a high voltage supply. Avoltage is then applied to the system. The applied voltage can be in the10 kV to 45 kV. The syringe pump can extrude the solution. The flow rateof the syringe pump can be in the range of 0.0001 uL/min to 423 mL/min.The collector plate can be static, rotating or moving in a specificlinear direction to give the fibers some directional orientation. Theshape of the collector plate can be varied with the collector platehaving but not limited to the following shapes: a flat surface, atextured surface, a curved surface, a square rod, a rectangular rod, around mandrel, an oval mandrel, a semi-circular mandrel or a combinationof these shapes. The distance of the needle tip to the collector platecan be altered. The distance of the needle tip to the collector platecan be in the 2-50 cm range. The collection plate can also be submergedin or sprayed with a solvent that assists in the precipitation of thenewly spun fibers. For example, an ethanol bath may be used during theelectrospinning of hyaluronic acid based derivatized polyhydric polymersof this disclosure.

The derivatized polyhydric polymers and/or compositions of thisdisclosure can be processed into the form of a fiber. A solution of aderivatized polyhydric polymer of the disclosure is prepared. Thissolution is then extruded through an orifice to produce a solventcontaining fiber. This fiber can be extruded into one or more solventbaths that assists in the formation of the fiber. The fiber is thendried to produce a solid fiber. The fibers can be prepared as amonofilament or a multifilament fiber. In one aspect, this fiber canthen be further processed through an annealing step. U.S. Pat. Nos.9,228,027, 5,520,916, 5,824,335, 8,389,498, US20130309494, US20150119783describe exemplary methods to produce fibers from a polysaccharide.These are incorporated by reference as means to produce fibers fromderivatized polyhydric polymers and/or compositions of this disclosure.

A fiber may be further processed by knitting or weaving, resulting in aknitted or woven composition. The knitted or woven composition can be inthe form of a mesh. The mesh can comprise a single derivatizedpolyhydric polymer and/or composition of this disclosure. In anotheraspect, the mesh can comprise 2 or more different derivatized polyhydricpolymers and/or compositions, of this disclosure. In another aspect, thefiber can be further processed into a braid. The braid can comprise asingle derivatized polyhydric polymer and/or composition of thisdisclosure. In another aspect, the braid can comprise 2 or moredifferent derivatized polyhydric polymers and/or compositions of thisdisclosure. For meshes or braids that use different derivatizedpolyhydric polymers and/or compositions of this disclosure, thederivatized polyhydric polymers and/or compositions used can result inthe mesh or braid having properties that change as a function of time.This includes degradation rates, water absorption, elongation, elasticmodulus, tensile strength, physical shape, lubricity, cell adhesion, ora combination of these properties.

The knitted, woven or braided derivatized polyhydric polymers and/orcompositions can be manufactured in the presence of a degradable ornon-degradable non-polysaccharide based material. These materialsinclude polyethylene, polypropylene, polyethylene terephthalate (PET),polytetrafluorethylene (PTFE), nylon, polyurethane, polyester,polyanhydride, polyorthoester, polycarbonate, poly-ester-co-carbonate),polyhydroxybutyrates or combinations thereof.

Crosslinked polymers of the present disclosure may take various physicalforms, including particle, film, lyophilized sponge, powder, particulate(e.g., milled, fragmented, precipitated and ground particulates), andmay be formed in-situe, e.g., spray or liquid.

A film of crosslinked derivatized polyhydric polymers and/orcompositions of this disclosure can be prepared by preparing a solutionof the derivatized polyhydric polymer and/or compositions to becrosslinked. The derivatized polyhydric polymer can be crosslinked byknown methods and/or those described herein. Prior to the finalcrosslinking process, the crosslinker is added, if required, and thesolution pH can be adjusted to initiate the crosslinking process. Thissolution can be then placed in a mold or drawn out on a surface, forexample, using a Gardner knife. The surface used can be glass, metalfoil, stainless steel, Teflon, nylon, polyethylene, polypropylene or arelease liner. The solution is then allowed to crosslink to form a gel.Heat can be applied to increase the rate of crosslinking. The solventcan then be removed to form the film.

A film of crosslinked derivatized polyhydric polymers of this disclosurecan be prepared by preparing a solution of the derivatized polyhydricpolymer to be crosslinked. The derivatized polyhydric polymer can becrosslinked by one of the methods described above. This solution can bethen placed in a mold or drawn out on a surface using a Gardner knife.The surface used can be glass, metal foil, stainless steel, Teflon,nylon, polyethylene, polypropylene, polystyrene, or a release liner. Thecrosslinking agent may be added prior to or following drying of thederivatized polyhydric polymer to form a crosslinked film or gel. Therate of residual solvent removal can be altered by using at least one ofthe following parameters: temperature, air or inert gas flow andpressure. To increase the rate of solvent evaporation, the temperaturecould be increased, the air or inert gas flow rate could be increased orthe pressure could be decreased. A combination of these process couldalso be used. To slow the rate of solvent evaporation, the temperaturecould be decreased, the air or inert gas flow rate could be reduced orthe pressure could be increased. A combination of these process couldalso be used. A film can comprise one of the derivatized polyhydricpolymers of this disclosure.

The films can also comprise two or more different derivatized polyhydricpolymers of this disclosure. A composite film can be prepared bypreparing a first film and then casting a second film on top of thefirst film. A composite film can be prepared by casting additionallayers sequentially on top of the previous layer. The layers of thecomposite film can comprise the same derivatized polyhydric polymer ifthe disclosure, different derivatized polyhydric polymers of thisdisclosure or a combination thereof. The films can comprise bothcrosslinked and non-crosslinked derivatized polyhydric polymers of thisdisclosure.

Lyophilized forms of the crosslinked derivatized polyhydric polymers ofthis disclosure can be prepared by making a solution of the derivatizedpolyhydric polymer, crosslinking the derivatized polyhydric polymer,freezing the crosslinked derivatized polyhydric polymer composition andthen placing the frozen derivatized polyhydric polymer composition undera vacuum such that the solvent is sublimed off to leave the resultingderivatized polyhydric polymer composition in the solid form. Alyophilized form of the derivatized polyhydric polymer of thisdisclosure can comprise one of the derivatized polyhydric polymers ofthis disclosure. In another aspect, the lyophilized form of thederivatized polyhydric polymer of this disclosure can comprise two ormore different derivatized polyhydric polymers of this disclosure. Theform of the lyophilized derivatized polyhydric polymer composition isdependent on the form of the container into which the solution waspoured and frozen. The form can be a rectangle, square, disk, triangle,trapezoid, rod or any other form in which a mold can be made. Thelyophilized derivatized polyhydric polymer compositions of thisdisclosure can comprise both crosslinked and non-crosslinked derivatizedpolyhydric polymers of this disclosure. In another aspect, thelyophilized derivatized polyhydric polymer composition, crosslinked ornon-crosslinked, can be rehydrated in the presence of other materialsdisclosed herein. In another aspect, a second lyophilization step may beperformed on a rehydrated derivatized polyhydric polymer composition.

In another aspect, the solution used to rehydrate the first lyophilizedderivatized polyhydric polymer, can be crosslinked. In another aspect,the derivatized polyhydric polymer composition produced from the secondcrosslinking step can be lyophilized to produce a dry porous derivatizedpolyhydric polymer composition. In another aspect, the derivatizedpolyhydric polymer solution may be directly combined with a biologicallyactive agent prior to lyophilization. In another aspect, the lyophilizedpolyhydric polymer composition s may be combined with a biologicallyactive agent through a rehydration process, which follows the firstlyophilization, which may or may not be followed by further drying.

Crosslinked derivatized polyhydric polymer compositions of thisdisclosure can be in the form of a powder or particulate. A powder orparticulate form can also be obtained through a milling, grinding, spraydrying or fragmentation process. Films, precipitated derivatizedpolyhydric polymers and/or compositions, dried derivatized polyhydricpolymers and/or compositions, lyophilized derivatized polyhydric polymerand/or compositions or derivatized polyhydric polymers and/orcompositions in dried in a form can be further process via a millingprocess (jet milling, roller milling, cryomilling, mechanical milling),a grinding or a fragmentation process. A combination of these processescan be used. Derivatized polyhydric polymer compositions with particlesize in the range of 100 nm to 5 mm can be prepared. Specific sizeranges of the powdered or particulate derivatized polyhydric polymercompositions of this disclosure can be prepared by separating thederivatized polyhydric polymer compositions according to size usingsieves. The distribution of particle sizes can be broad with a standarddeviation of the average size of greater than 40%. The distribution ofparticle sizes can be narrow with a standard deviation of the averagesize of less than 30%. The final powdered or particulate form of thederivatized polyhydric polymers and/or compositions of this disclosurecan comprise a single distribution of average particle sizes or it cancomprise two or more distributions of particles prepared by mixingparticles of different average particle size.

The crosslinked derivatized polyhydric polymers compositions of thisdisclosure can be formed into a solid form by preparing a solution ofthe derivatized polyhydric polymer in a solvent that can be removed,pouring this solution into a mold of a specific shape, crosslinking thederivatized polyhydric polymer in the mold, and then removing thesolvent such that a solid form of the crosslinked derivatized polyhydricpolymer composition is obtained. The molds used can be of various shapesand can include but are not limited to cubes, rectangles, rods,semi-circular rode and tubes. The solid derivatized polyhydric polymercomposition of this disclosure can then be removed from the mold.

In one aspect, the derivatized polyhydric polymers of this disclosurecan be used to prepare an in-situ gel forming composition. A derivatizedpolyhydric polymer of this disclosure that contains available vinylsulfone groups can be reacted with a compound that contains at least twoavailable free thiol groups or a compound that contains at least 2available amine groups, preferably primary or secondary amines.Illustrative thiol containing compounds include PEG-dithiol (HS-PEG-SH),3-arm PEG-tri-thiol (glycerine core), 4-arm PEG-tetrathiol(pentaerythritol core), or 8-arm PEG-octa-thiol (hexaglycerine core).The foregoing multi-armed PEG reagents may also have fewer than all armsfunctionalized with thiol. Additional suitable thiol reagents having PEGas the central molecule are available from Laysan Bio (Arab, Ala.), aswell as aromatic dithiols such as those available from NanoScience.Other suitable thiol crosslinking agents include dimercaptosuccinicacid, 2,3-dimercapto-1-propanesulfonic acid, dihydrolipoic acid,peptides containing at least 2 cysteine amino acids, a thiolfunctionalized polysaccharide, thiol functionalized dextran, andthiol-functionalized hyaluronic acid. In one aspect, the derivatizedpolyhydric polymers of this invention can be prepared as a solution.This solution can be mixed with either a solution of the thiolcontaining compound or the solid form of the thiol containing compoundto produce the gel composition.

The derivatized polyhydric polymers of the present disclosure, e.g., thefirst, second and third derivatives of a starting polymer, may be incombination with one or more other derivatized polyhydric polymers orother components, such as pharmaceutically acceptable excipients, orother known or common components of compositions. Thus, the presentdisclosure provides compositions comprising derivatized polyhydricpolymers of the present disclosure.

The derivatized polyhydric polymers and compositions thereof of thisdisclosure can be used to treat living organisms. These living organismsinclude humans, animals, birds, fish, insects and plants. Thederivatized polyhydric polymers and compositions thereof used in theindications described below can comprise, non-crosslinked derivatizedpolyhydric polymers, crosslinked derivatized polyhydric polymers or acombination thereof. In another aspect, the derivatized polyhydricpolymer compositions used can comprise only one of the derivatizedpolyhydric polymers of this disclosure. In another aspect, thederivatized polyhydric polymer compositions used can comprise two ormore of the derivatized polyhydric polymers of this disclosure. Thederivatized polyhydric polymers and compositions thereof can furthercomprise one or more excipients. The derivatized polyhydric polymers andcompositions thereof can further comprise one or more biologicallyactive agents. The derivatized polyhydric polymers and compositionsthereof that are used in the indications described below can be in asterile form. Sterilization can be attained through sterile filtration,aseptic manufacture, gamma radiation, e-beam radiation, ethylene oxide,dry heat, autoclaving, or a combination thereof.

For instance, the derivatized polyhydric polymer compositions of thisdisclosure can also comprise an excipient. The excipient may be apharmaceutically acceptable excipient. Excipients that can be usedinclude but are not limited to natural polymers, synthetic polymers,thermosreversible polymers, biodegradable polymers, buffers, complexingagents, tonicity modulators, ionic strength modifiers, solvents,anti-oxidants, preservatives, viscosity modifiers, pH modifiers,surfactants, emulsifiers, phospholipids, stabilizers and porogens.

Excipient polymers that can be used include but are not limited tosodium alginate, calcium alginate, dextran, carboxymethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, hyaluronic acid, hyaluronic acid derivatives, dextran,heparin, chitosan, chitosan acetate, chitosan lactate, chitin, xanthamgum, Xylan, guar gum, pullulan, locust bean gum, starch, gelatin,collagen, derivatized collagen, and acacia (gum Arabic).

Excipient degradable polymers that can be used include but are notlimited to polyesters, polyether esters, polyorthoesters, poly estercarbonates, polycarbonates, polyanhydrides, polyhydroyalkonate (e.g.Polyhydroxybutyrate, polyhydroxyvalerates), polyurethanes, poly esterurethanes. The polymers can be in the form of linear, branched, or starshaped. The polymers can be initiated from compounds that us a singlepoint of initiation, two points of initiation, 3 points of initiation,four points of initiation, 6 points of initiation or 8 points ofinitiation. Polymers can include but are not limited to polymers thatare comprise repeat units derived from at least one of the followingmonomers: l-lactide, dl-lactide, glycolide, trimethylene carbonate,epsilon-caprolactone, p-dioxanone and a morpholinedione

Excipient synthetic polymers that can be used include but are notlimited to polyacrylic acid and salts thereof, polyvinylpyrollidone,Pluronics 127, pluronics F68, polyethylene glycol, polyethylene oxide,polyvinyl alcohol.

Complexing agents can include but are not limited to α-cyclodextrin,β-cyclodextrin (2-Hydroxypropyl)-Beta-Cyclodextrin, Sulfobutylether BetaCyclodextrin Sodium, Ethylenediaminetetraacetic acid (EDTA)

Phospholipids that can be used include but are not limited tohydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol,L-α-dimyristoylphosphatidylcholine, L-α-dimyristoylphosphatidylglycerol

Surfactants that can be used include ionic and non-ionic surfactants.Ionic surfactants can include cationic, anionic and zwitterionicsurfactants. Non-ionic surfactants can include but are not limited to(Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-_-tocopherolpolyethylene glycol 1000 succinate, Brij, Myrj, polysorbate 20,polysorbate 80, polysorbate 40, polysorbate 60, polysorbate 65,polysorbate 85, Solutol HS 15, sorbitan monooleate (Span 80), Sorbitanmonopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitantrioleate (Span 8) poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS,Labrasol, Gellucire 44/14, nonoxynol-9, Softigen 767, octylbeta-D-glycopyranoside (OGP), hexyl beta-D-glucopyranoside (HGP), Octylbeta-D-1-thioglucopyranoside (TGP), Decyl-beta-D-glucopyranoside (DGP),Dodecyl-beta-D-glucopyranoside (DdGP), N-octyl beta-D-Maltoside (ODM),decyl beta-D-maltopyranoside (DMP), cyclohexyl-ethanoyl-maltoside,n-decyl- and n-dodecyl-sucrose, and mono- and di-fatty acid esters ofPEG 300, 400, or 1750. Anionic surfactants can include but are notlimited to sodium lauryl sulfate, fatty acid salts, sodium laurethsulfate, dioctyl sodium sulfosuccinate. Cationic surfactants can includebut are not limited to Phosphatidylcholine (Lecithin), cetrimide,cetrimonium bromide, benethonium chloride, dimethyldioctadecyl ammoniumchloride, tetradecyl trimethyl ammonium bromide, cetylpyridiniumchloride, esterquat, and benzalkonium chloride. Zwiterionic surfactantscan include but are not limited to Cocamidopropyl betaine,(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) andcocamidopropyl hydroxysultaine, phosphatidylserine,phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins.

Solvents that can be used include water-soluble organic solvents.Water-soluble organic solvents include but are not limited topolyethylene glycol 200, polyethylene glycol 300, polyethylene glycol400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone,dimethylacetamide, and dimethylsulfoxide.

Tonicity modifiers that can be used include but are not limited todextrose, sucrose, mannitol, glycerin, sodium chloride, and potassiumchloride.

pH modifiers that can be used include but are not limited to citric acidand its salts, salts of phosphoric acid, tartatic acid, lactic acid,glycolic acid, sodium hydroxide, phosphoric acid, sulfuric acid, oxalicacid and hydrochloric acid.

Anti-oxidants that can be used include but are not limited to ascorbicacid, butylated hydroxyanisole, Butylhydroxytoluene, Vitamin A, vitaminE, α-tocopherol, thioglycerol, cysteine, acetylcysteine, cystine,dithioerythreitol, dithiothreitol, glutathione, Sodium bisulfite, Sodiummetabisulfite, thiourea, uric acid, melatonin, propyl gallate, tertiarybutylhydroquinone and combinations thereof.

Emulsifiers that can be used include but are not limited to GlycerylMonostearate, Isopropyl Palmitate, Polyethylene Glycol 400 Monostearate,as well as the compounds listed as surfactants and combinations thereof.

Preservatives that can be used include but are not limited to benzoicacid, sorbic acid, boric acid, methylparaben, ethylparaben,propylparaben, butylparaben, sodium benzoate, sodium propionate, phenylethyl alcohol, chlorobutanol, benzyl alcohol, potassium sorbate, phenol,chlorocresol, o-phenyl phenol, thiomersal, nitromersol, phenylmercuricnitrate, phenylmercuric acetate, benzalkonium and combinations thereof.

The excipients can include at least one solvent. The solvents used caninclude but are not limited to water, ethanol, dimethylsulfoxide, ethyllactate, ethyl acetate, benzyl alcohol, benzyl benzoate, triacetin,N-methylpyrrolidone, 2-pyrrolidone, propylene carbonate, polyethyleneglycol (PEG200), polyethylene glycol (PEG400), glycofurol andcombinations thereof.

Buffers that can be used include aqueous solutions prepared using one ormore of the following: potassium hydrogen phthalate, sodium hydrogenphthalate, potassium or sodium dihydrogen phosphate, dipotassium ordisodium hydrogen phosphate, phosphoric acid, boric acid, sodiumacetate, acetic acid, ammonium chloride, ammonium acetate,(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), citric acid andsodium citrate.

In another aspect, the derivatized polyhydric polymer compositions ofthis disclosure can further comprise an inorganic compound. Theinorganic compounds that can be used include but are not limited tobarium sulfate, calcium hydroxylapatite or hydroxyapatite, tricalciumphosphate (TCP) [including the various forms, for example α-TCP, β-TCP,and Biphasic Tricalcium Phosphate (BCP)], calcium phosphate and calciumsulphate.

In one aspect, the derivatized polyhydric polymers of this disclosurecan be prepared as a composition that comprises one or more excipients.In another aspect, the derivatized polyhydric polymers of thisdisclosure can be suspended in a composition that comprises one or moreexcipients. In another aspect, the derivatized polyhydric polymers ofthis disclosure can be rehydrated in a composition that comprises one ormore excipients. In another aspect, the derivatized polyhydric polymersof this disclosure can be prepared as separate compositions that cancomprise one or more excipients with the separate solutions being mixedprior to use. In another aspect, the derivatized polyhydric polymers ofthis disclosure can be prepared in the presence of one or moreexcipients and then converted to a solid form by one or more of themethods described in this disclosure.

Compositions of the present disclosure may comprise a biologicallyactive agent in addition to a derivatized polyhydric polymer asdescribed herein and optionally other components. Exemplary biologicallyactive agents include, without limitation, small molecule drugs,peptides, proteins, growth factors, hormones, antibodies, agonists,antagonists, anti-bacterial and/or anti-fungal agents.

Biologically active agents that can be incorporated into formulationswith the compositions described include: antiandrogens, antibacterial,antioestrogens, androgens and anabolic agents, antibiotics, antimigrainedrugs, antihistamines, antianxiety drugs, antidiuretics, antihistamines,antirheumatoid agents, antigens, analgesics, antidepressants,antiinflammatories, anesthetics, aminoglycosides antibodies, antiviral,adrenergic stimulants, anticonvulsants, antiangina agents,antiarrhyrthmics, antimalarials, anti-mitotic, anthelmintics, anoreticagents, antitussives, antipruritics, antipyretics, anti-alzheimer'sagents, anti-Parkinson's agents, antiemetics and antinauseants,antihypertensives, anticoagulants, antifungals, antimicrobials,allergens, antidiarrheals, antihyperuricaemia agents, adrenergicstimulants, antiparasitic agents, antiproliferative agents,antipsychotic drugs, antithyroid agents, beta-adrenergic blockingagents, bronchodilators; bronchospasm relaxants, blood clotting factors,blood coagulation factors, cytotoxic agents, cytostatic agents,chemotherapeutics, clot inhibitors, clot dissolving agents, cells, CNSstimulants, Corticosteroids, calcium channel blockers, cofactors,ceramides, cardiotonic glycosides, cytokines (e.g., lymphokines,monokines, chemokines); colony stimulating factors (e.g., GCSF, GM-CSF,MCSF); dermatological agents, decongestants, diuretics, expectorants,endectocide agents, growth factors, hemostatic agents, hypoglycemicagents, hormones and hormone analogs, hypercalcemia, Hypnotics,interleukins (IL-2, IL-3, IL-4, IL-6); interferons (.beta.-IFN,.alpha.-IFN and .gamma.-IFN); immunosuppressants, muscle relaxants,microorganisms, non-steroidal anti-inflammatory agents, nucleic acids,nutritional agents, neuromuscular blocking agents, neuroleptics,Neurotoxins, nutraceuticals, oligonucleotides, oestrogens, obstetricdrugs, ovulation inducers, opioids, progestogens, pituitary hormones,Pituitary inhibitors proteins, peptides, polysaccharides, proteaseinhibitors, prostaglandins, quinolones, reductase inhibitors, sulfadrugs, sclerosant, sedatives, sodium channel blockers, steroids,steroidal anti-inflammatory agents, smoking cessation agents, toxins,thrombolytic agents, thyroid hormones, tumor necrosis factor; vesicles,vitamins, viruses, vasodilators, vaccines

Additional representative examples of biologically active agents thatmay be suitable for use in the compositions of the present disclosureinclude, but are not limited to: Antidiarrheals such as diphenoxylate,loperamide and hyoscyamine; Antihypertensives such as hydralazine,minoxidil, captopril, enalapril, clonidine, prazosin, debrisoquine,diazoxide, guanethidine, methyldopa, reserpine, trimethaphan; Calciumchannel blockers such as diltiazem, felodipine, amlodipine,nitrendipine, nifedipine and verapamil; Antiarrhyrthmics such asamiodarone, flecainide, disopyramide, procainamide, mexiletene andquinidine, Antiangina agents such as glyceryl trinitrate, erythrityltetranitrate, pentaerythritol tetranitrate, mannitol hexanitrate,perhexilene, isosorbide dinitrate and nicorandil; Beta-adrenergicblocking agents such as alprenolol, atenolol, bupranolol, carteolol,labetalol, metoprolol, nadolol, nadoxolol, oxprenolol, pindolol,propranolol, sotalol, timolol and timolol maleate; Cardiotonicglycosides such as digoxin and other cardiac glycosides and theophyllinederivatives; Adrenergic stimulants such as adrenaline, ephedrine,fenoterol, isoprenaline, orciprenaline, rimeterol, salbutamol,salmeterol, terbutaline, dobutamine, phenylephrine, phenylpropanolamine,pseudoephedrine and dopamine; Vasodilators such as cyclandelate,isoxsuprine, papaverine, dipyrimadole, isosorbide dinitrate,phentolamine, nicotinyl alcohol, co-dergocrine, nicotinic acid, glycerltrinitrate, pentaerythritol tetranitrate and xanthinol;Antiproliferative agents such as paclitaxel, estradiol, actinomycin D,sirolimus, tacrolimus, everolimus, 5-fluorouracil and dexamethasone;Antimigraine preparations such as ergotanmine, dihydroergotamine,methysergide, pizotifen and sumatriptan; Anticoagulants and thrombolyticagents such as warfarin, dicoumarol, low molecular weight heparins suchas enoxaparin, streptokinase and its active derivatives; Hemostaticagents such as aprotinin, tranexamic acid and protamine; Analgesics andantipyretics including the opioid analgesics such as buprenorphine,dextromoramide, dextropropoxyphene, fentanyl, alfentanil, sufentanil,hydromorphone, methadone, morphine, oxycodone, papaveretum, pentazocine,pethidine, phenopefidine, codeine, dihydrocodeine; acetylsalicylic acid(aspirin), paracetamol, synthetic alpha2-adrenoreceptor agonist,dexmedetomidine hydrochloride, flunixin meglumine, meperidine,phenylbutazone and phenazone; Immunosuppressants, antiproliferatives andcytostatic agents such as rapamycin (sirolimus) and its analogs(everolimus and tacrolimus); Neurotoxins such as capsaicin, botulinumtoxin (botox); Hypnotics and sedatives such as the barbituratesamylobarbitone, butobarbitone and pentobarbitone and other hypnotics andsedatives such as chloral hydrate, chlormethiazole, hydroxyzine andmeprobamate; Antianxiety agents such as the benzodiazepines alprazolam,bromazepam, chlordiazepoxide, clobazam, chlorazepate, diazepam,flunitrazepam, flurazepam, lorazepam, nitrazepam, oxazepam, temazepamand triazolam; Neuroleptic and antipsychotic drugs such as thephenothiazines, chlorpromazine, fluphenazine, pericyazine, perphenazine,promazine, thiopropazate, thioridazine, trifluoperazine; andbutyrophenone, droperidol and haloperidol; and other antipsychotic drugssuch as pimozide, thiothixene and lithium; Antidepressants such as thetricyclic antidepressants amitryptyline, clomipramine, desipramine,dothiepin, doxepin, imipramine, nortriptyline, opipramol, protriptylineand trimipramine and the tetracyclic antidepressants such as mianserinand the monoamine oxidase inhibitors such as isocarboxazid, phenelizine,tranylcypromine and moclobemide and selective serotonin re-uptakeinhibitors such as fluoxetine, paroxetine, citalopram, fluvoxamine andsertraline; CNS stimulants such as caffeine and 3-(2-aminobutyl) indole;Antipruritics can include compounds such as synthetic Janus Kinase (JAK)inhibitors, NK-1 receptor antagonists, antibodies that neutralizeinterleukin-31 (IL-31). These can include oclacitinib maleate,Serlopitant, and Lokivetmab, Anti-alzheimer's agents such as tacrine;Anti-Parkinson's agents such as amantadine, benserazide, carbidopa,levodopa, benztropine, biperiden, benzhexol, procyclidine and dopamine-2agonists such as S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0923),Anticonvulsants such as phenytoin, valproic acid, primidone,phenobarbitone, methylphenobarbitone and carbamazepine, ethosuximide,methsuximide, phensuximide, sulthiame and clonazepam, Antiemetics andantinauseants such as the phenothiazines prochloperazine,thiethylperazine, a neurokinin (NK1) receptor antagonist, maropitantcitrateand 5HT-3 receptor antagonists such as ondansetron andgranisetron, as well as dimenhydrinate, diphenhydramine, metoclopramide,domperidone, hyoscine, hyoscine hydrobromide, hyoscine hydrochloride,clebopride and brompride; Non-steroidal anti-inflammatory agentsincluding their racemic mixtures or individual enantiomers whereapplicable, preferably which can be formulated in combination withdermal and/or mucosal penetration enhancers, such as ibuprofen,flurbiprofen, ketoprofen, aclofenac, diclofenac, aloxiprin, aproxen,aspirin, diflunisal, fenoprofen, indomethacin, mefenamic acid, naproxen,phenylbutazone, piroxicam, salicylamide, salicylic acid, sulindac,desoxysulindac, tenoxicam, tramadol, ketoralac, flufenisal, salsalate,triethanolamine salicylate, aminopyrine, antipyrine, oxyphenbutazone,apazone, cintazone, flufenamic acid, clonixerl, clonixin, meclofenamicacid, 6-chloro-α-methyl-9H-carbazole-2-acetic acid (carprofen),flunixin, coichicine, demecolcine, allopurinol, oxypurinol, benzydaminehydrochloride, dimefadane, indoxole, intrazole, mimbane hydrochloride,paranylene hydrochloride, tetryda mine, benzindopyrine hydrochloride,fluprofen, ibufenac, naproxol, fenbufen, cinchophen, diflumidone sodium,fenamole, flutiazin, metazamide, letimide hydrochloride, nexeridinehydrochloride, octazamide, molinazole, neocinchophen, nimazole,proxazole citrate, tesicam, tesimide, tolmetin, and triflumidate;Antirheumatoid agents such as penicillamine, aurothioglucose, sodiumaurothiomalate, methotrexate and auranofin; Muscle relaxants such asbaclofen, diazepam, cyclobenzaprine hydrochloride, dantrolene,methocarbamol, orphenadrine and quinine; Agents used in gout andhyperuricaemia such as allopurinol, colchicine, probenecid andsulphinpyrazone; Oestrogens such as estradiol, oestriol, estrone,ethinylestradiol, mestranol, stilbestrol, dienestrol, epiestriol,estropipate and zeranol; Progesterone and other progestagens such asallylestrenol, dydrgesterone, lynestrenol, norgestrel, norethyndrel,norethisterone, norethisterone acetate, gestodene, levonorgestrel,medroxyprogesterone and megestrol; Antiandrogens such as cyproteroneacetate and danazol; Antioestrogens such as tamoxifen and epitiostanoland the aromatase inhibitors, exemestane and 4-hydroxy-androstenedioneand its derivatives; Androgens and anabolic agents such as testosterone,methyltestosterone, clostebol acetate, drostanolone, furazabol,nandrolone oxandrolone, stanozolol, trenbolone acetate,dihydro-testosterone, 17-(.alpha.-methyl-19-noriestosterone andfluoxymesterone; 5-alpha reductase inhibitors such as finasteride,turosteride, LY-191704 and MK-306; Corticosteroids such asbetamethasone, betamethasone valerate, cortisone, dexamethasone,dexamethasone 21-phosphate, fludrocortisone, flumethasone, fluocinonide,fluocinonide desonide, fluocinolone, fluocinolone acetonide,fluocortolone, halcinonide, halopredone, hydrocortisone, hydrocortisone17-valerate, hydrocortisone 17-butyrate, hydrocortisone 21-acetate,methylprednisolone, prednisolone, prednisolone 21-phosphate, prednisone,triamcinolone, triamcinolone acetonide; Glycosylated proteins,proteoglycans, glycosaminoglycans such as chondroitin sulfate; chitin,acetyl-glucosamine, hyaluronic acid; Complex carbohydrates such asglucans; Further examples of steroidal anti-inflammatory agents such ascortodoxone, fludroracetonide, fludrocortisone, difluorsone diacetate,flurandrenolone acetonide, medrysone, amcinafel, amcinafide,betamethasone and its other esters, chloroprednisone, clorcortelone,descinolone, desonide, dichlorisone, difluprednate, flucloronide,flumethasone, flunisolide, flucortolone, fluoromethalone, fluperolone,fluprednisolone, meprednisone, methylmeprednisolone, paramethasone,cortisone acetate, hydrocortisone cyclopentylpropionate, cortodoxone,flucetonide, fludrocortisone acetate, flurandrenolone, aincinafal,amcinafide, betamethasone, betamethasone benzoate, chloroprednisoneacetate, clocortolone acetate, descinolone acetonide, desoximetasone,dichlorisone acetate, difluprednate, flucloronide, flumethasonepivalate, flunisolide acetate, fluperolone acetate, fluprednisolonevalerate, paramethasone acetate, prednisolamate, prednival,triamcinolone hexacetonide, cortivazol, formocortal and nivazol;Pituitary hormones and their active derivatives or analogs such ascorticotrophin, thyrotropin, follicle stimulating hormone (FSH), aGonadotropin-releasing hormone (GnRH) analog, u deslorelin acetate,cetrorelix acetate, Gonadorelin acetate, clomiphene, Human chorionicgonadotropin (HCG), luteinizing hormone (LH) and gonadotrophin releasinghormone (GnRH); Hypoglycemic agents such as insulin, chlorpropamide,glibenclamide, gliclazide, glipizide, tolazamide, tolbutamide andmetformin; Thyroid hormones such as calcitonin, thyroxine andliothyronine and antithyroid agents such as carbimazole andpropylthiouracil; Other miscellaneous hormone agents such as octreotide;Pituitary inhibitors such as bromocriptine; Ovulation inducers such asclomiphene; Diuretics such as the thiazides, related diuretics and loopdiuretics, bendrofluazide, chlorothiazide, chlorthalidone, dopamine,cyclopenthiazide, hydrochlorothiazide, indapamide, mefruside,methycholthiazide, metolazone, quinethazone, bumetanide, ethacrynic acidand frusemide and potasium sparing diuretics, spironolactone, amilorideand triamterene; Antidiuretics such as desmopressin, lypressin andvasopressin including their active derivatives or analogs; Obstetricdrugs including agents acting on the uterus such as ergometrine,oxytocin and gemeprost; Prostaglandins such as alprostadil (PGE1),prostacyclin (PGI2), dinoprost (prostaglandin F2-alpha) and misoprostol;Antimicrobials including the cephalosporins such as cephalexin,cefoxytin and cephalothin; Penicillins such as amoxycillin, amoxycillinwith clavulanic acid, ampicillin, bacampicillin, benzathine penicillin,benzylpenicillin, carbenicillin, cloxacillin, methicillin,phenethicillin, phenoxymethylpenicillin, flucloxacillin, meziocillin,piperacillin, ticarcillin and azlocillin; Tetracyclines such asminocycline, chlortetracycline, tetracycline, demeclocycline,doxycycline, methacycline and oxytetracycline and othertetracycline-type antibiotics; Amnioglycoides such as amikacin, amikinsulfate, gentamicin, kanamycin, neomycin, netilmicin and tobramycin;Antifungals such as amorolfine, isoconazole, clotrimazole, econazole,miconazole, nystatin, terbinafine, bifonazole, amphotericin,griseofulvin, ketoconazole, fluconazole and flucytosine, salicylic acid,fezatione, ticlatone, tolnaftate, triacetin, zinc, pyrithione and sodiumpyrithione; Quinolones such as nalidixic acid, cinoxacin, ciprofloxacin,enoxacin and norfloxacin; Sulphonamides such as phthalysulphthiazole,sulfadoxine, sulphadiazine, sulphamethizole and sulphamethoxazole;Sulphones such as dapsone; Other miscellaneous antibiotics such aschloramphenicol, clindamycin, erythromycin, erythromycin ethylcarbonate, erythromycin estolate, erythromycin glucepate, erythromycinethylsuccinate, erythromycin lactobionate, roxithromycin, lincomycin,natamycin, nitrofurantoin, spectinomycin, vancomycin, aztreonarn,colistin IV, metronidazole, tinidazole, secnidazole, ornidazole, fusidicacid, trimethoprim, and 2-thiopyridine N-oxide; halogen compounds,particularly iodine and iodine compounds such as iodine-PVP complex anddiiodohydroxyquin, hexachlorophene; chlorhexidine; chloroaminecompounds, silver sulfadiazine, silver, nanoparticulate silver, silvernitrate, silver zeolites, silver cations, AgPO3 Ag3PO4, Ag4P2O7,exsalt®SD7 (Exciton Technologies) exsalt®T7 (Exciton Technologies);Lincomycin Hydrochloride, tricyclic tetrahydroquinoline antibacterialagents, 8-pyrazinyl-S-spiropyrimidinetrione-oxazinoquinolinederivatives, 3-spiropyrimidinetrione-quinoline derivatives,thiadiazol-spiropyrimidinetrione-quinoline derivatives,(2R,4S,4aS)-10-fluoro-2,4-dimethyl-8-(4-methyloxazol-2-yl)-2,4,4a,6-tetra-hydro-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(-3′H)-trione,(2R,4S,4aS)-9,10-difluoro-2,4-dimethyl-8-(3-methylisoxazol-5-yl)-2,4,4a,6-tetrahydro-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,-4′,6′(3′H)-trione,(2R,4S,4aS)-10-fluoro-2,4-dimethyl-8-(oxazol-2-yl)-2,4,4a,6-tetrahydro-1H-,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-tri-one,(2R,4S,4aS)-9,10-difluoro-2,4-dimethyl-8-(2-methyloxazol-5-yl)-2,4,4a,6t-etrahydro-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′-,6′(3′H)-trione,(2R,4S,4aS)-9,10-difluoro-2,4-dimethyl-8-(oxazol-4-yl)-2,4,4a,6-tetrahydr-o-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione,(2R,4S,4aS)-9-fluoro-2,4-dimethyl-8-(4-methyloxazol-2-yl)-2,4,4a,6-tetrah-ydro-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3-′H)-trione,(2R,4S,4aS)-9,10-difluoro-8-(4-(4-fluorophenyl)oxazol-5-yl)-2,4-dimethyl-2,4,4a,6-tetrahydro-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5’-pyrimidine]-2′,4′,6′(3′H)-trione,(2S,4R,4aR)-2,4-dimethyl-8-(oxazol-5-yl)-2,4,4a,6-tetrahydro-1H,1′H-spiro-[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione,(2S,4R,4aR)-8-(4-ethyloxazol-2-yl)-9,10-difluoro-2,4-dimethyl-2,4,4a,6-te-trahydro-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,-6′(3′H)-trione,(2R,4S,4aS)-9,10-difluoro-2,4-dimethyl-8-(oxazol-2-yl)-2,4,4a,6-tetrahydr-o-1H,1′H-spiro[[1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione,benzoyl peroxide; Antituberculosis drugs such as ethambutol, isoniazid,pyrazinamide, rifampicin and clofazimine; Antimalarials such asprimaquine, pyrimethamine, chloroquine, hydroxychloroquine, quinine,mefloquine and halofantrine; compounds such as Azithromycin, Aztreonam,Cefaclor, Cefadroxil, Cefazolin, Cefdinir, Cefepime Hydrochloride,(cefoperazone sodium, Ceftaroline fosamil, avibactam, Ceftazidimesodium, Ceftibuten, ceftiofur, Tazobactam, cefovecin sodium[(6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-8-oxo-3-[(2S)-tetrahydro-2-furanyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid, monosodium salt] Cefuroxime Axetil, Cefuroxime, Cephalexin,Chloramphenicol Sodium, Ciprofloxacin HCl, Clarithromycin, Clindamycinhydrochloride, Clindamycin Palmitate hydrochloride,Clindamycinphosphate, Dalbavancin Hydrochloride, Daptomycin, Demeclocyclinehydrochloride, Dicloxacillin, Doripenem, Doxycycline, Doxycyclinecalcium, Doxycycline hyclate, Doxycycline monohydrate, Ertapenem sodium,Erythromycin, Erythromycin Ethylsuccinate, Erythromycin lactobionate,Erythromycin stearate, Erythromycin, Fosfomycin tromethamine,Gemifloxacin mesylate, Gentamicin Sulfate, Imipenem, Kanamycin,Levofloxacin, Lincomycin hydrochloride, Linezolid, Meropenem,Methenamine Hippurate, Metronidazole, Metronidazole , Micafungin sodium,Minocycline Hydrochloride, Minocycline, Moxifloxacin hydrochloride,Nafcillin, Nalidixic acid, Neomycin Sulfate, Nitrofurantoin,Norfloxacin, Ofloxacin, Oritavancin diphosphate, Oxacillin, PenicillinG, Penicillin G benzathine, Penicillin G Sodium, Penicillin V Potassium,Piperacillin Sodium, Polymyxin B Sulfate, Quinupristin, dalfopristin,Spectinomycin hydrochloride, Streptomycin, Sulfamethoxazole, TedizolidPhosphate, Telavancin, Telithromycin, Tetracycline Hydrochloride,Ticarcillin disodium, Tigecycline, Tobramycin Sulfate, Tobramycin,Trimethoprim hydrochloride, tulathromycin, Vancomycin hydrochloride.

Antiviral agents may be included in the compositions of the presentdisclosure, where exemplary antiviral agents include acyclovir andacyclovir prodrugs, famcyclovir, zidovudine, didanosine, stavudine,lamivudine, zalcitabine, saquinavir, indinavir, ritonavir, n-docosanol,tromantadine and idoxuridine. Other suitable biologically active agentsinclude Anthelmintics such as mebendazole, thiabendazole, niclosamide,praziquantel, pyrantel embonate and diethylcarbamazine; Cytotoxic agentssuch as plicamycin, cyclophosphamide, dacarbazine, fluorouracil and itsprodrugs (described, for example, in International Journal ofPharmaceutics, 111, 223-233 (1994)), methotrexate, procarbazine,6-mercaptopurine and mucophenolic acid; Anorectic and weight reducingagents including dexfenflurarnine, fenfluramine, diethylpropion,mazindol and phentermine; Agents used in hypercalcaemia such ascalcitriol, dihydrotachysterol and their active derivatives or analogs;Antitussives such as ethylmorphine, dextromethorphan and pholcodine;Antiparasitic and Endectocide agents such as moxidectin, Ivermectin,Niclosamide, Praziquantel, Pyrantel, Pyrvinium, Albendazole,Flubendazole, Mebendazole, Thiabendazole

Compositions of the present disclosure may include: an expectorant suchas carbolcysteine, bromihexine, emetine, quanifesin, ipecacuanha andsaponins; Decongestants such as phenylephrine, phenylpropanolamine andpseudoephedrine; Bronchospasm relaxants such as ephedrine, fenoterol,orciprenaline, rimiterol, salbutamol, sodium cromoglycate, cromoglycicacid and its prodrugs (described, for example, in International Journalof Pharmaceutics 7, 63-75 (1980)), terbutaline, ipratropium bromide,salmeterol and theophylline and theophylline derivatives; Antihistaminessuch as meclozine, cyclizine, chlorcyclizine, hydroxyzine,brompheniramine, chlorpheniramine, clemastine, cyproheptadine,dexchlorpheniramine, diphenhydramine, diphenylamine, doxylamine,mebhydrolin, pheniramine, tripolidine, azatadine, diphenylpyraline,methdilazine, terfenadine, astemizole, loratidine and cetirizine; Localanaesthetics such as benzocaine, bupivacaine, amethocaine, lignocaine,lidocaine, cocaine, cinchocaine, dibucaine, mepivacaine, prilocaine,etidocaine, veratridine (specific c-fiber blocker) and procaine; Stratumcorneum lipids, such as ceramides, cholesterol and free fatty acids, forimproved skin barrier repair [Man, et al. J. Invest. Dermatol., 106(5),1096, (1996)]; Neuromuscular blocking agents such as suxamethonium,alcuronium, pancuronium, atracurium, gallamine, tubocurarine andvecuronium; sclerocing agents or sclerosants may be a surfactant or itmay be selected from the group consisting of ethanol, dimethylsulfoxide, sucrose, sodium chloride, dextrose, glycerin, minocycline,tetracycline, doxycycline, polidocanol, sodium tetradecyl sulfate,sodium morrhuate, and sotradecol. an angiogenesis inhibitor; a5-lipoxygenase inhibitor or antagonist; a chemokine receptor antagonist;a cell cycle inhibitor; a taxane; an anti-microtubule agent; paclitaxel;an analogue or derivative of paclitaxel; a vinca alkaloid; camptothecinor an analogue or derivative thereof; a podophyllotoxin, wherein thepodophyllotoxin may be an etoposide or an analogue or derivativethereof; an anthracycline, wherein the anthracycline may be doxorubicinor an analogue or derivative thereof or the anthracycline may bemitoxantrone or an analogue or derivative thereof; a platinum compound;a nitrosourea; a nitroimidazole; a folic acid antagonist; a cytidineanalogue; a pyrimidine analogue; a fluoropyrimidine analogue; a purineanalogue; a nitrogen mustard or an analogue or derivative thereof; ahydroxyurea; a mytomicin or an analogue or derivative thereof; an alkylsulfonate; a benzamide or an analogue or derivative thereof; anicotinamide or an analogue or derivative thereof; a halogenated sugaror an analogue or derivative thereof; a DNA alkylating agent; ananti-microtubule agent; a topoisomerase inhibitor; a DNA cleaving agent;an antimetabolite; a nucleotide interconversion inhibitor; ahydroorotate dehydrogenase inhibitor; a DNA intercalation agent; an RNAsynthesis inhibitor; a pyrimidine synthesis inhibitor; a cyclindependent protein kinase inhibitor; an epidermal growth factor kinaseinhibitor; an elastase inhibitor; a factor Xa inhibitor; afarnesyltransferase inhibitor; a fibrinogen antagonist; a guanylatecyclase stimulant; a heat shock protein 90 antagonist; which may be ageldanamycin or an analogue or derivative thereof; a guanylate cyclasestimulant; a HMGCoA reductase inhibitor, which may be simvastatin or ananalogue or derivative thereof; an IKK2 inhibitor; an IL-1 antagonist;an ICE antagonist; an IRAK antagonist; an IL-4 agonist; animmunomodulatory agent; sirolimus or an analogue or derivative thereof;everolimus or an analogue or derivative thereof; tacrolimus or ananalogue or derivative thereof; biolmus or an analogue or derivativethereof; tresperimus or an analogue or derivative thereof; auranofin oran analogue or derivative thereof; 27-0-demethylrapamycin or an analogueor derivative thereof; gusperimus or an analogue or derivative thereof;pimecrolimus or an analogue or derivative thereof; ABT-578 or ananalogue or derivative thereof; an inosine monophosphate dehydrogenase(IMPDH) inhibitor, which may be mycophenolic acid or an analogue orderivative thereof or 1-.alpha.-25 dihydroxy vitamin D.sub.3 or ananalogue or derivative thereof; a leukotriene inhibitor; an MCP-1antagonist; an MMP inhibitor; an NF kappa B inhibitor, which may be Bay11-7082; an NO antagonist; a p38 MAP kinase inhibitor, which may be SB202190; a phosphodiesterase inhibitor; a TGF-.beta. inhibitor; athromboxane A2 antagonist; a TNF-alpha-antagonist; a TACE inhibitor; atyrosine kinase inhibitor; vitronectin inhibitor; a fibroblast growthfactor inhibitor; a protein kinase inhibitor; a PDGF receptor kinaseinhibitor; an endothelial growth factor receptor kinase inhibitor; aretinoic acid receptor antagonist; a platelet derived growth factorreceptor kinase inhibitor; a fibrinogen antagonist; an antimycoticagent; sulconizole; a bisphosphonate; a phospholipase A1inhibitor; ahistamine H1/H2/H3 receptor antagonist; a macrolide antibiotic; aGPIIb/IIIa receptor antagonist; an endothelin receptor antagonist; aperoxisome proliferator-activated receptor agonist; an estrogen receptoragent; a somastostatin analogue; a neurokinin 1 antagonist; a neurokinin3 antagonist; a VLA-4 antagonist; an osteoclast inhibitor; a DNAtopoisomerase ATP hydrolyzing inhibitor; an angiotensin I convertingenzyme inhibitor; an angiotensin II antagonist; an enkephalinaseinhibitor; a peroxisome proliferator-activated receptor gamma agonistinsulin sensitizer; a protein kinase C inhibitor; a ROCK (rho-associatedkinase) inhibitor; a CXCR3 inhibitor; Itk inhibitor; a cytosolicphospholipase A.sub.2-.alpha. inhibitor; a PPAR agonist; animmunosuppressant; an Erb inhibitor; an apoptosis agonist; a lipocortinagonist; a VCAM-1 antagonist; a collagen antagonist; an .alpha.-2integrin antagonist; a TNF-.alpha. inhibitor; a nitric oxide inhibitor;and a cathepsin inhibitor. anti-fibrin and fibrinolytic agents,including plasmin, streptokinase, single chain urokinase, urokinase,t-PA (tissue type plasminogen activator), aminocaproic acid;anti-platelet agents including, aspirin, prostacyclins (and analogues);glycoprotein IIb/IIIa agents including monoclonal antibodies, peptides(e.g. ReoPro, Cilastagel, eptifibatide, tirofiban, ticlopidine,Vapiprost, dipyridamole, forskolin, angiopeptin, argatroban),thromboxane inhibitors; anti-thrombin and anti-coagulant agents,including dextan, heparin, LMW heparin (Enoxaparin, Dalteparin),hirudin, recombinant hirudin, anti-thrombin, synthetic antithrombins,thrombin inhibitors, Warfarin (and other coumarins); anti-mitotic,antiproliferative and cytostatic agents, including vincristine,vinblastine, paclitaxel, methotrexate, cisplatin, fluorouracil,rapamycin, azathioprine, cyclophosphamide, mycophenolic acid,corticosteroids, colchicine, nitroprusside; antiangiogenic andangiostatic agents, including paclitaxel, angiostatin and endostatin;genetic materials, DNA, DNA sequences, polynucleotides, andoligonucleotides; ACE inhibitors (e.g. Cilazapril, Lisinopril,Captopril); growth factor (e.g. VEGF, FGF) antagonists; antioxidants andvitamins (e.g. Probucol, Tocopherol); calcium channel blockers (e.g.nifedipine); fish oil (omega 3-fatty acid); phosphodiesterase inhibitors(e.g. dipyridamole); nitric acid donor (e.g. Molsidomine); somatostatinanalogues (e.g. angiopeptin); immunosuppresives and anti-inflammatoryagents (e.g. prednisolone, glucocorticoid and dexamethasone);antimicrobials (e.g. rifamycin) and radionuclides, including alpha, betaand gamma emitting isotopes (e.g. Re-188, Re-186, I-125, Y-90); COX-2inhibitors such as Celecoxib and Vioxx; kinase inhibitors, such asepidermal growth factor kinase inhibitor, tyrosine kinase inhibitors,MAP kinase inhibitors protein transferase inhibitors, Resten-NG, smokingcessation agents such as nicotine, bupropion and ibogaine; Insecticidesand other pesticides which are suitable for local application;Dermatological agents, such as vitamins A, C, B1, B2, B6, B 12, B12.alpha., and E, vitamin E acetate and vitamin E sorbate; Allergens fordesensitisation such as house, dust or mite allergens; Nutritionalagents and neutraceuticals, such as vitamins, essential amino acids andfats; Macromolecular pharmacologically active agents such as proteins,enzymes, peptides, polysaccharides (such as cellulose, amylose, dextran,chitin), nucleic acids, cells, tissues, and the like; Bone mendingbiochemicals such as calcium carbonate, calcium phosphate, tricalciumphosphate, hydroxyapetite or bone morphogenic protein (BMP); Angiogenicgrowth factors such as Vascular Endothelial Growth Factor (VEGF) andepidermal growth factor (EFG), cytokines interleukins, fibroblasts andcytotaxic chemicals; and Keratolytics such as the alpha-hydroxy acids,glycolic acid and salicylic acid; and DNA, RNA or otheroligonucleotides. Vaccines that contain Hendra virus (HeV) Gglycoprotein and/or Nipah virus G glycoprotein, Lutenising HormoneReleasing Hormone (LHRH) peptide, LHRH-diphtheria toxoid conjugate,porcine circovirus type 2 (PCV2) antigen, a porcine reproductive andrespiratory syndrome virus antigen, Mycoplasma hyopneumoniae proteinantigen. Proteins or protein fragments, for example ORFI Torque tenovirus protein, or other TTV proteins or fragments, antigens againstAeromonas salmonicida, antigens against Vibrio anguillarum, and antigensagainst V. salmonicida. Growth factors include but are not limited toVascular Endothelial Growth Factor (VEGF) and epidermal growth factor(EFG), Growth Differentiation Factors (GDFs), Fibroblast Growth Factors(FGF-1 through FGF-23), Osteoprotegerin, Cartilage Derived MorphogenicProteins (CDMPs, which can be a foundation for soft or hard tissue), LimMineralization Proteins (LMPs)Interleukins (IL-1 through IL-13),Insulin-like Growth Factor-1, Connective Tissue Growth Factor (CTGF),platelet derived growth factor (PDGF), nerve growth factors, neutrophinsBrain-derived neurotrophic factor (BDNF), Nerve growth factor (NGF),Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4)], Transforming growthfactors (TGF-α, TGF-β), Tumor necrosis factor (TNF). Growth factorAgonists or antagonists as well as antibodies against these growthfactors. Biologically active agents that can be used to treat maculardegeneration include but are not limited to bevacizumab and ranibizumab

In one aspect, compositions of the present disclosure are formulatedfor, and are useful for, wound healing. Compositions may be formulatedfor suitable administration, e.g., nasal or topical administration.Compositions may include one or more suitable biologically active agentsfor wound healing. The wounds treated can include but are not limited todiabetic ulcers, burns, pressure wounds, abrasions, incisions, cornealabrasion, incisions following ocular surgery, blisters, damaged tissuefollowing sinus surgery, abdominal surgery, tendon repair or jointrepair.

For example, in one aspect, derivatized polyhydric polymer compositionsof the disclosure can be in the form of dry particles. In anotheraspect, the derivatized polyhydric polymer compositions of thedisclosure can be in the form of a lyophilized derivatized polyhydricpolymer compositions. In another aspect, the derivatized polyhydricpolymer compositions of the disclosure can be on the form of a non-wovenderivatized polyhydric polymer compositions. In one aspect, thenon-woven derivatized polyhydric polymer compositions can be produced byan electrospinning process. In another aspect, the derivatizedpolyhydric polymer of the disclosure can be in the form of a film.Compositions can be packaged directly or indirectly in a foil pouch tominimize moisture absorption during storage.

Derivatized polyhydric polymer compositions of the disclosure can beapplied directly to a wound site. The derivatized polyhydric polymercompositions can absorb exudate from the wound. Once sufficient exudateis absorbed, the dry derivatized polyhydric polymer compositions willturn into a gel. In another aspect, the derivatized polyhydric polymercompositions of the disclosure further comprise water or saline suchthat a gel is obtained. In one aspect, the gel can be applied directlyto the wound.

In one aspect, the derivatized polyhydric polymer compositions of thedisclosure, once applied to the wound, can be covered by a have amoisture retaining semi-permeable film. The film can further comprise anadhesive that will retain the film at the site of application. Themoisture retaining semi-permeable adhesive film can be made from apolyurethane or a silicone material with an adhesive coating on at leastthe border or edges of the film. In one aspect, the adhesive can be anacrylic based adhesive. The semi-permeable film is permeable to oxygenand carbon dioxide, as well as water vapor but will prevent bacterialtransmission.

In another aspect, the derivatized polyhydric polymers and compositionsthereof of the disclosure can be applied to a semi-permeable film suchthat the product is premade and ready to use in that the derivatizedpolyhydric polymer compositions of the disclosure and the semipermeablefilm are a single unit. Compositions can be packaged directly orindirectly in a foil pouch. In one aspect, the derivatized polyhydricpolymer of the disclosure comprises a hyaluronic acid that has beenderivatized with sulfonate groups, where such a derivatized polyhydricpolymer may be used, for example, in a composition intended for woundhealing.

In another aspect, the derivatized polyhydric polymers and compositionsof the disclosure can be used as bulking agents. These bulking agentscan be used to treat stress urinary incontinence, fecal incontinence,Gastroesophageal Reflux Disease (GERD), prostate-rectum spacer forreduction in rectal damage as a result of radiation treatment forprostate cancer. In one aspect, the injected derivatized polyhydricpolymer compositionscan be in the form of derivatized polyhydric polymerthat may or may not comprise crosslinks.

In one aspect, the derivatized polyhydric polymers and compositions ofthe disclosure can be used as a dermal filler to fill voids, defects andto treat moderate to severe wrinkles and folds. Derivatized polyhydricpolymer compositions can be injected as a solution or suspension. In oneaspect, at least one derivatized polyhydric polymer in a derivatizedpolyhydric polymer composition is crosslinked. In one aspect, thecrosslinked derivatized polyhydric polymer of this disclosure used inthe dermal filler composition has a hyaluronidase (or correspondingpolysaccharide degrading enzyme for other polysaccharides) degradationrate that is the same as or less than that of polyhydric polymer (e.g.,hyaluronic acid) that is not derivatized. The derivatized polyhydricpolymers and compositions can be used treat areas where dermaldepressions, wrinkles or scars are found including, but not limited tonasolabial folds, forehead, furrow lines and vertical lip lines. Inanother aspect, the derivatized polyhydric polymers and compositions canbe used for lip augmentation and breast augmentation.

In another aspect, the derivatized polyhydric polymers and compositionsused as dermal fillers may comprise a drug (e.g., biologically activeagent) to reduce pain associated with the procedure. As used herein, abiologically active agent includes compounds or molecules that may bereferred to as a drug. Such compounds include benzocaine, bupivacaine,amethocaine, lignocaine, lidocaine, cocaine, cinchocaine, dibucaine,mepivacaine, prilocaine, etidocaine, veratridine (specific c-fiberblocker) and procaine. In another aspect, the derivatized polyhydricpolymers and compositions used as dermal fillers may comprise adegradable water-insoluble polymer (e.g. polyester such as PLGA, PLLAetc), a water insoluble non-degradable polymer (e.g.polymethylmethacrylate [PMMA]) or inorganic material (e.g. calciumhydroxyapatite). In another aspect, the derivatized polyhydric polymersand compositions used as dermal fillers are in the form of particles ofa crosslinked hydrogel. In one aspect, median size (Dv50) of theparticles are in the range of 100 μm to 800 μm. In another aspect, themedian size (Dv50) of the particles are in the range of 200 μm to 600μm. In one aspect, the crosslinked hydrogel particles are suspended in asaline solution. In another aspect, the hydrogel particles are suspendedin a solution of hyaluronic acid oa hyaluronic acid derivative of thisdisclosure. In one aspect, the crosslinked hydrogel suspension is in aprefilled syringe in which the contents of the syringe are sterile. Inanother aspect, the hydrogel particle suspension is injectable throughat least a 27 G needle.

In one aspect, the derivatized polyhydric polymers and compositions asdisclosed herein are formulated for, and are useful for,viscosupplementation. The derivatized polyhydric polymers may or may notcrosslinked, and the compositions may optionally contain a biologicallyactive agent.

Visco supplementation is the process of injecting a derivatizedpolyhydric polymer composition into the joint to relieve pain. In thepreferred aspect, the polyhydric polymer is hyaluronic acid or aderivative thereof. Derivatized polyhydric polymer compositions can beinjected into one or more joint spaces of the body. Suitable jointsinclude, but are not limited to, knee, shoulder, ankle, elbow, hip,trapeziometacarpal joint, finger joint, wrist joints, temporomandibularjoint, back and neck. In another aspect, the derivatized polyhydricpolymers used can comprise crosslinked derivatized polyhydric polymers.The derivatized polyhydric polymer compositions can comprise one or moreexcipients or diluents. The derivatized polyhydric polymer compositionsof the disclosure that can be used for osteoarthritis treatment can beinjected through a needle of between 18 gauge and 21 gauge. Derivatizedpolyhydric polymer composition of the disclosure can comprise abiologically active agent. In one aspect, the biologically active agentcan be, but is not limited to, a corticosteroid, a local anesthetic, anantibody, a peptide or an anti-inflammatory compound or molecule. Thevolume of the solution that comprises the derivatized polyhydric polymercomposition of the disclosure can range from 0.5 ml to 10 mL with thepreferred aspect being in the 2 mL to 6 mL for injection into the knee.In one aspect, crosslinked derivatized polyhydric polymer hydrogelparticles are suspended in a saline solution. In another aspect, thederivatized polyhydric polymer particles are suspended in a solution ofhyaluronic acid or a hyaluronic acid derivative on this disclosure. Inone aspect, the crosslinked derivatized polyhydric polymer suspension isin a prefilled syringe in which the contents of the syringe are sterile.

In one aspect, the derivatized polyhydric polymers and compositions asdisclosed herein are formulated for, and are useful for, adhesionprevention. The derivatized polyhydric polymers may or may notcrosslinked, and the compositions may optionally comprise a biologicallyactive agent. Areas of the body where methods of treatment for adhesionprevention is wanted include spinal and abdominal areas, particularlyafter surgical procedures, as a coating on dura substitute, in nasalprocedures or devices, in conjunction with ear, elbow, and tendonmedical procedures. Exemplary biologically active agents include, butare not limited to, anti-inflammatory and pain medicines.

In one aspect, the derivatized polyhydric polymers of this disclosuremay be used to reduce the incidence and severity of adhesions and scartissue that may occur following injury or a surgical procedure. Theseadhesions can include abdominal adhesions, pelvic adhesions, heartadhesions, joint adhesions, tendon adhesions (e.g. flexor tendon,Achilles tendon, patella tendon), spinal adhesions, lumbar adhesions,nerve adhesions, dural adhesions, sinus adhesions. The derivatizedpolyhydric polymer compositions can further comprise one or moreexcipient. The derivatized polyhydric polymer compositions of thedisclosure can further comprise a biologically active agent. In oneaspect, the biologically active agent can be, but is not limited to, acorticosteroid, a local anesthetic, an antibody, a peptide or ananti-inflammatory. In one aspect, the derivatized polyhydric polymer ofthis disclosure is derived from hyaluronic acid or a hyaluronic acidderivative. In one aspect, the derivatized polyhydric polymer can be inthe form of a crosslinked hydrogel. In another aspect, the derivatizedpolyhydric polymer of the disclosure can be in a crosslinked form thathas been lyophilized to form a porous foam or it could be as a solid orperforated film.

In one aspect, the derivatized polyhydric polymers and compositions asdisclosed herein are formulated for, and are useful for, tissue sealing.The derivatized polyhydric polymers may or may not crosslinked, and thecompositions may optionally comprise a biologically active agent.

In one aspect, the derivatized polyhydric polymer of the disclosure thatcontains residual vinyl sulfone groups can be reacted with a compoundthat has 2 or more free thiol functional groups such that a crosslinkedderivatized polyhydric polymer is produced. In one aspect, thederivatized polyhydric polymer of the disclosure that contains freevinyl sulfone groups can be prepared as a solution. In one aspect, thesolution can be prepared using saline. In one aspect, the derivatizedpolyhydric polymer of the disclosure that contains residual vinylsulfone groups can be prepared as a first solution and the derivatizedpolyhydric polymer that has 2 or more free thiol functional groups canbe prepared as a second solution. The pH of either the first or thesecond solution can be adjusted such that the pH of the solution isgreater than pH 8. This can be accomplished by using a solution that hasa pH of greater than 8 to dissolve either the derivatized polyhydricpolymer of the disclosure that contains residual vinyl sulfone groups orthe compound that has 2 or more free thiol functional groups, addingbuffer components to either the derivatized polyhydric polymer of thedisclosure that contains residual vinyl sulfone groups or to thecompound that has 2 or more free thiol functional groups.

In one aspect, the first and second solution can be combined and appliedto the tissue surface resulting in a mixture. In one aspect, the mixturecan be applied through a needle or canula. In another aspect, themixture can be applied using a spray applicator. Examples of sprayapplicators include but are not limited to the Fibrijet SA-3674 andSA-3675 (Nordson Medical, 261 Cedar Hill Street, Marlborough, Mass.01752, United States). In another aspect, the mixture can be appliedusing a gas assisted spray applicator. Examples of gas assisted sprayapplicators include but are not limited to the Fibrijet SA-3651 andSA-3652, (Nordson Medical, 261 Cedar Hill Street, Marlborough, Mass.01752, United States).

In one aspect, the derivatized polyhydric polymer composition can beapplied to the tissue in a liquid form and after 3 minutes thederivatized polyhydric polymer composition is in a gel form. The timerequired to convert from the liquid form to the gel form depends on thespecific application. In one aspect the liquid to gel conversion cantake less than 2 minutes. In another aspect the liquid to gel conversioncan take less than 30 seconds. In another aspect, the liquid to gelconversion can take less than 15 seconds.

The derivatized polyhydric polymer composition for tissue sealing mayfurther comprise an excipient. The derivatized polyhydric polymercomposition can further comprise a biologically active agent.

In one aspect, the derivatized polyhydric polymer compositions of thisdisclosure are combined with a biologically active agent to treatbacterial vaginosis. The derivatized polyhydric polymer compositions ofthis disclosure can be formulated such that the derivatized polyhydricpolymer compositions is tissue adhesive and adheres to the vaginaltissue for a period of greater than 2 hours. The derivatized polyhydricpolymer compositions can further comprise one or more excipient. Thederivatized polyhydric polymer compositions of the disclosure canfurther comprise a biologically active agent. In an aspect, thebiologically active agent can be an antibacterial agent. In an aspect,the antibacterial can be, but is not limited to, clindamycin,tinidazole, metronidazole, secnidazole and ornidazole. Formulationscomprising the derivatized polyhydric polymers of this disclosure, canbe applied intravaginally.

In one aspect, the derivatized polyhydric polymers and compositions ofthe disclosure are selected to provide ocular application. For example,eye drops for dry eyes/lubricating eye drops for contact lenses.

In one aspect, derivatized polyhydric polymer compositions of thisdisclosure can be used as eye drops. The eye drops can be used to treatdry eyes, a disease of the eye, infected ocular tissue, inflamed oculartissue, as a lubricant for the surface of the eye, as a lubricant foruse with contact lenses and to assist in healing of the eye followingtrauma or a surgical procedure to the eye or surrounding tissue.Surgical procedures to the eye can include but are not limited tocataract surgery, intra-ocular lens replacement, fixing detatchedretinas, tumor removal, glaucoma surgery, refractive surgery, cornealsurgery, vitreo-retinal surgery, eye muscle surgery, oculoplasticsurgery, surgery involving the lacrimal punctum, canaliculus, and sac.An ocular formulation comprising derivatized polyhydric polymers of thisdisclosure can further comprise an excipient. The derivatized polyhydricpolymers of this disclosure can be formulated into a solution orsuspension which is then administered to the eye. An ocular formulationcomprising derivatized polyhydric polymers of this disclosure canfurther comprise a biologically active agent. The biologically activeagent can be present as part of the solution or it can be in the form ofa suspension or emulsion. The derivatized polyhydric polymers of thisdisclosure can be formulated into a solution or suspension which is thenadministered to the eye.

In another aspect, derivatized polyhydric polymer compositions of thisdisclosure can be prepared to be used to lubricate and wet contactlenses. The contact lens can be immersed prior to use or could be storedin a solution that contains derivatized polyhydric polymers of thisdisclosure. The solution can comprise one or more excipients. Thesolution can further comprise boric acid or sodium borate. The solutioncan be formulated to be preservative free.

In one aspect, derivatized polyhydric polymers of this disclosure can beformed into a formulation that is inserted into the lacrimal punctum,the lacrimal canaliculus or the lacrimal sac. Derivatized polyhydricpolymers of the disclosure can be in the form of a solution, swollenhydrogel or a dehydrated hydrogel. In one aspect, the derivatizedpolyhydric polymer compositionscan further comprise an excipient. Inanother aspect, the derivatized polyhydric polymer is crosslinked. Inanother aspect, derivatized polyhydric polymer compositions furthercomprise a biologically active agent. In one aspect, the biologicallyactive agent can be but is not limited to a corticosteroid (for example,dexamethasone, mometasone fuorate, triamcinolone acetonide,triamcinolone hexacetonide, triamcinolone acetate, betamethasone,fluoromethalone, hydrocortisone, medrysone or prednisolone),prostaglandins (for example, latanoprost, travoprost or bimatoprost),beta blockers (for example timolol or betaxolol), alpha-adrenergicagonists (for example apraclonidine or brimonidine), carbonic anhydraseinhibitors (for example dorzolamide or brinzolamide), mitotic orchlorinergic agents (for example pilocarpine)

In another aspect, a derivatized polyhydric polymer and/or compositionsis crosslinked in the presence of a biologically active agent and thendried. In another aspect, a derivatized polyhydric polymer iscrosslinked, dried, reswollen in the presence of a biologically activeagent and then dried. In another aspect, the biological agent isincorporated into the uncrosslinked derivatized polyhydric polymer insolution. In another aspect, the derivatized polyhydric polymer isdried, reswollen in the presence of a biologically active agent and thendried. Any of the dried formulations can be of suitable dimensions suchthat it can be placed in the lacrimal punctum. Upon contact withlachrymal fluid and tears, the final dried formulation hydrates, andswells in such a manner as to be physically retained in the punctum. Inanother aspect, the dried formulation can be inserted into thecanaliculus. Upon contact with lachrymal fluid and tears, the driedformulation hydrates, and swell in such a manner as to be physicallyretained in the canaliculus. The formulation could then release thecontained biologically active agent over a period of 24 hours to 3weeks. In one aspect, the biologically active agent is released in asustained manner for a period of 7 days. In one aspect, the biologicallyactive agent is released in a sustained manner for a period of 4 weeks.In an aspect, the dried formulation can be inserted intravitreally sothat the biologically active agent is delivered into the vitreous of theeye. In an aspect, the dried formulation is inserted into the anteriorchamber of the eye.

In an aspect, the derivatized polyhydric polymers and compositions ofthe disclosure are selected to provide a punctal plug. The punctal plugmay comprise a biologically active agent, e.g., steroid or a pain reliefdrug.

In one aspect, the derivatized polyhydric polymer compositions of thisdisclosure can be used to treat mucositis. Examples of mucositis includeoral and vaginal mucositis. During cancer treatments, the rapidlydivided epithelial cells lining the gastro-intestinal tract (which goesfrom the mouth to the anus) break down leaving the mucosal tissue opento ulceration and infection. This leads to mucocitis. Oral mucositis canoften occur following chemotherapy and radiation treatments. It can leadpain and increased risk of infection. This can lead to nutritionalproblems due to these symptoms reducing the ability and desire to eat.Providing a coating that covers these lesions, can reduce the pain andpotential for infection. The derivatized polyhydric polymers of thisdisclosure can be formulated such that the derivatized polyhydricpolymer compositions is tissue adhesive and adheres to the mucosaltissue of the mouth tissue or the vagina for a period of greater than 2hours. The derivatized polyhydric polymer compositions can furthercomprise one or more excipients. The derivatized polyhydric polymercompositions of the disclosure can further comprise a biologicallyactive agent. In one aspect, the biologically active agent can be, butis not limited to, a local anesthetic, an anti-infective, ananti-inflammatory or a combination thereof. Local anesthetics caninclude but are not limited to benzocaine, bupivacaine, amethocaine,lignocaine, lidocaine, cocaine, cinchocaine, dibucaine, mepivacaine,prilocaine, etidocaine, veratridine (specific c-fiber blocker) andprocaine. For oral mucositis, the derivatized polyhydric polymercompositions of the disclosure can be formulated such that it can beapplied as an oral rinse or applied as a gel. For vaginal mucositis, thederivatized polyhydric polymer compositions of the disclosure can beformulated such that it can be applied intravaginally to the vaginaltissue surface.

In one aspect, derivatized polyhydric polymer compositions of thisdisclosure can be used to treat a surgical site during and followingcanalplasty, tympanoplasty, myringoplasty, stapedectomy mastoidprocedures, or any other procedure related to the ear. Derivatizedpolyhydric polymer compositionscan be used to modulate wound healing aswell as to control bleeding. The derivatized polyhydric polymercompositions of this disclosure can be in the form of a lyophilizedsponge, an electrospun matrix, a film, a gel or a combination of theseforms. The derivatized polyhydric polymer compositions of the disclosurecan comprise an excipient. In another aspect, the derivatized polyhydricpolymer compositions of the disclosure can comprise a biologicallyactive agent.

In another aspect, the derivatized polyhydric polymer compositions ofthe disclosure can be used to treat otitis media, acute otitis externa,balance disorders (for example Meniere' disease, tinnitus andsensorineural hearing loss. Derivatized polyhydric polymer compositionsof this disclosure can be in the form of a solution, a suspension, alyophilized sponge, an electrospun matrix, a film, a gel, a solidrod-like form, or a combination of these forms. Derivatized polyhydricpolymer compositions of the disclosure can comprise an excipient. Inanother aspect, a derivatized polyhydric polymer compositions of thedisclosure can comprise a biologically active agent. To treat infectionsof the ear, the derivatized polyhydric polymer can comprise anantibiotic, an antibacterial, an antiviral, an antifungal or acombination thereof. In one aspect, derivatized polyhydric polymercompositionscomprising at least one biologically active agent include,but are not limited to, amoxicillin, clavulanate, cefuroxime axetil,ceftriaxone, Levofloxacin, a cephalosporin, atrimethoprim-sulfamethoxazole, a macrolide, ofloxacin, gentamicinsulfate, tobramycin sulfate and ciproflaxin, In another aspect,derivatized polyhydric polymer compositionscan comprise acorticosteroid. Corticosteroids can include but are not limited tobetamethasone, betamethasone valerate, cortisone, dexamethasone,dexamethasone 21-phosphate, fludrocortisone, flumethasone, fluocinonide,fluocinonide desonide, fluocinolone, fluocinolone acetonide,fluocortolone, halcinonide, halopredone, hydrocortisone, hydrocortisone17-valerate, hydrocortisone 17-butyrate, hydrocortisone 21-acetate,methylprednisolone, prednisolone, prednisolone 21-phosphate, prednisone,triamcinolone, triamcinolone acetonide, mometasone fuorate. In anotheraspect, a combination of an antibiotic and a corticosteroid can be addedto the derivatized polyhydric polymer compositions of the disclosure. Inone aspect, the derivatized polyhydric polymer compositions of thedisclosure can be applied to the area to be treated by being appliedwith a dropper, a syringe, through a needle or catheter or by physicallyplacing a derivatized polyhydric polymer compositions.

In one aspect, the derivatized polyhydric polymer compositions of thedisclosure can comprise a biologically active agent. The derivatizedpolyhydric polymers of this disclosure can be used as a matrix fromwhich the biologically active agent can be delivered. In one aspect therelease profile of the biologically active agent into a phosphatebuffered saline solution if slower than that of the normal dissolutionprofile of the biologically active agent. In one aspect, the derivatizedpolyhydric polymer compositions of the disclosure can be in the form ofa crosslinked gel.

In one aspect, the treatment using the drug delivery formulation can bea single injection or could be two or more injections that are separatedby a period of time. The composition can be injected subcutaneously,intra-dermally or intra-muscularly. The derivatized polyhydric polymercompositionscan be injected through a needle, trocar, catheter, tube, orcanula.

In another aspect, the contents of the prefilled syringe or vial aresterile. In another aspect, the contents of the prefilled syringe orvial are stable at 2-8° C. or 20-25° C. for at least 6 months,preferably 12 months and most preferably 24 months. In another aspect,the drug delivery formulation can be applied topically or byinstillation.

In one aspect, the derivatized polyhydric polymers of this disclosure ina crosslinked form can be used as device to plug a defect following theremoval of a piece of tissue or the needle track following a biopsyprocedure. In another aspect, a crosslinked form of the derivatizedpolyhydric polymer compositions can be prepared and then dried. Thedried derivatized polyhydric polymer compositions can be delivered intothe needle track or the site that a piece of tissue was removed. Thedried derivatized polyhydric polymer compositions can absorb moisturefrom the surrounding tissue to rehydrate and swell such that the swollensize is larger than the initial size of the derivatized polyhydricpolymer compositions. The swollen derivatized polyhydric polymercomposition is then retained at the site into which it is placed. Inanother aspect, the crosslinked dried derivatized polyhydric polymercomposition can be used to seal a hole in the tissue where thecrosslinked derivatized polyhydric polymer composition is placed in thehole and it swells to seal off the hole. An example of this could be toseal lung tissue following puncturing of the lung following a biopsy orsurgical procedure. In another aspect, the crosslinked dried derivatizedpolyhydric polymer compositioncan comprise an element, such as a metalpiece that is visible under x-ray or fluoroscopy examination. The metalpiece can take on various forms such as but not limited to a flat piece,a rod, a coil, a loop, a hoop, hook, a number and a letter of thealphabet. In one aspect, the crosslinked dried derivatized polyhydricpolymer composition can comprise a biologically active agent. In anotheraspect, the biologically active agent can have hemostatic properties. Inone aspect, the crosslinked dried derivatized polyhydric polymercomposition can comprise collagen, chitosan or thrombin.

In one aspect, the derivatized polyhydric polymers and compositions ofthe present disclosure are formulated for, and are useful for, a plugfor female sterilization. Female sterilization can be accomplished byinserting a plug into the fallopian tube. This plug can provide aphysical barrier to the passage of the ovum into the uterus as well asto the sperm reaching the ovum. The predominant procedure to effectfemale sterilization in a laparoscopic procedure in which the fallopiantubes are severed and then ligated. In other versions of the procedure,the fallopian tubes can be closed using clips or rings to clamp thenclosed. Cauterization has also been used to seal the fallopian tubes.These procedures are generally classed as major surgery, usuallyrequires general anesthesia and the patient requires a recuperationperiod. Transvaginal sterilization procedures were an alternative to thelaproscopic procedures as they were less invasive. Initial transvaginalprocedures used chemical agents, such as sodium morrhuate, orquinacrine, methyl cyanoacrylate and silver nitrate, but the successrates and side effects have limited their use. Hysteroscopic tubalsterilization has emerged as a minimally invasive alternative toconventional tubal ligation. Hysteroscopic tubal sterilization can beperformed in approximately 10 minutes in an office setting without theuse of general or even local anesthesia.

Two hysteroscopic tubal sterilization products were commercialized, butboth have been removed from the US market by the end of 2018. The Essuresystem consisted of a device insert that is loaded into a single-usedelivery system. The device consisted of an inner coil of stainlesssteel and polyethylene terephthalate (PET) fibers and an outer coil ofnickel-titanium (nitinol). The metal components hold the device in placewhile the PET fibers allow tissue ingrowth into the device which willlead to occlusion of the fallopian tube. This ingrowth process does taketime and so the patient must use other forms of contraception for 3months. At this stage a hysterosalpingogram is performed to confirmplacement and tubal occlusion. The device is permanent and remains inthe patient for the rest of the patient's life. This product received ablack box warning over potential safety concerns, and was subsequentlyremoved from the market in the US. The device had previously beenremoved from the market overseas.

Another sterilization method was developed by Hologic. The Adiana®sterilization method used radiofrequency energy to cause controlledthermal damage of the lining of the fallopian tube lumen. Following thethermal injury to the fallopian tube, a porous non-degradable siliconeplug is placed in the thermally injured fallopian tube. Over a fewweeks, tissue ingrowth into the porous plug results in occlusion of thefallopian tube. A hysterosalpingogram is performed at 3 months toconfirm tubal occlusion. The silicone plug is a permanent implant. TheAdiana® system has been withdrawn from the market.

The Essure system and the Adiana® system both leave a permanent devicein the patient. This can potentially lead to longer term safety issuesfor the patient. Having a system that comprises a degradable plugcomponent would be beneficial in that little to no derivatizedpolyhydric polymer and/or composition will remain permanently within thepatient. The method and devices described herein provide a means toocclude the fallopian tube that will result in a reduction in theability of a female to become pregnant. The method involves mechanicallyinjuring the lining of the fallopian tube followed by the insertion of adegradable plug.

A method for mechanically injuring the fallopian tube is to insert adevice that comprises a rough surface into the fallopian tube and thenphysically move the device in a rotational motion, a linear motion thatfollows the fallopian tube or a combination thereof. This motion can berepeated more than once. This physical movement is continued until theendothelial layer of the fallopian tube where the physical motion occursis either partially removed or completely removed.

The device used to denude the endothelial layer of the fallopian tubecan comprise a series of fiber radiating from a central core. In oneaspect this device is similar in structure to a bottle brush, e.g., arod with bristles (fibers) extending perpendicularly from the rod.

In one aspect, the fibers can be spaced evenly apart in a continuousmanner. In one aspect, the fibers can be in rows with spaces between therows. In one aspect, the fibers could be oriented in a spiral shapealong the axis of the device. In one aspect, the fibers can be orientedin one or more linear rows that are aligned about parallel with the axisfrom which they emanate. In another aspect, the fibers are in one ormore rows such that the rows are about perpendicular to the axis fromwhich they emanate.

In one aspect, the fibers can be made from a non degradable polymer. Thepolymers that can be used to prepare the fibers include but are notlimited to polyethylene, polypropylene, polyethylene terephthalate(PET), nylon, polyurethane, polyetheretherketone (PEEK),polyaryletherketone (PAEK), fluorocarbon polymers such aspolytetrafluoroethylene, silk and combinations thereof.

In one aspect, the fibers can be made from a metal. The metals that canbe used to prepare the fibers include but are not limited to stainlesssteel, titanium, nitinol, magnesium, alloys of Co—Cr—Mo, Cr—Ni—Cr—Mo,CP—Ti, Ti—Al—V, Ti—Al—Nb, Ti-13Nb-13Zr, Ti—Mo—Zr—Fe or combinationsthereof.

In one aspect, the central core (rod) of the denuding device cancomprise a core prepared from the twisting of 2 or more metal strandstogether such that the fibers are trapped between the twisted metalstrands. The metals that can be used to prepare the central core includebut are not limited to stainless steel, titanium, nitinol, magnesium,alloys of Co—Cr—Mo, Cr—Ni—Cr—Mo, CP—Ti, Ti—Al—V, Ti—Al—Nb, Ti-13Nb-13Zr,Ti—Mo—Zr—Fe or combinations thereof.

In one aspect, the terminal end of the central core (rod) that is firstintroduced into the fallopian tube can comprise an atraumatic tip thatdoes not damage the tissue as the device is being guided into thedesired location in the fallopian tube. This atraumatic tip can be arounded end cap, a domed shaped end, a cone shaped end with a roundedtip. The surface of the atraumatic tip can have a smooth surface. Theatraumatic tip can be made of a non-degradable polymer or a metal. Thenon-degradable polymers that can be used to manufacture the atraumatictip include but are not limited to polyethylene, polypropylene,polyethylene terephthalate (PET), nylon, polyurethane,polyetheretherketone (PEEK), polyaryletherketone (PAEK), fluorocarbonpolymers such as polytetrafluoroethylene, silk and combinations thereof.The metals that can be used to prepare the atraumatic tip include butare not limited to stainless steel, titanium, nitinol, magnesium, alloysof Co—Cr—Mo, Cr—Ni—Cr—Mo, CP—Ti, Ti—Al—V, Ti—Al—Nb, Ti-13Nb-13Zr,Ti—Mo—Zr—Fe or combinations thereof.

The atraumatic tip can be attached to the central core by a crimpingprocess, a molding process, a process that uses an adhesive to bond thetip to the central core, or a thermal process.

The plug can comprise a hydrogel. In one aspect, the hydrogel isprepared using one or more crosslinked derivatized polyhydric polymerand/or compositions of this disclosure. A hydrogel comprising apolyhydric polymer composition in the form a rod that is larger than thesize of the fallopian tube is prepared. The hydrogel rod is then dried.The hydrogel can be dried at normal atmospheric pressures or underreduced atmospheric pressure. In one aspect, the hydrogel can belyophilized. Once delivered to the desired site, the hydrogel plug wouldabsorb moisture from the fallopian tube and swell. The swelling of thehydrogel plug will enable the hydrogel plug to be retained at the sitewhere it was placed.

In one aspect, the hydrogel further comprises a porogen to facilitatethe formation of pores within the hydrogel. The porogen can compriseparticulates. The particulates can comprise a degradable polymer.Degradable polymers that can be used as porogens include but are notlimited to degradable polyesters, polyanhydrides, polyurethanes,polyether-esters, polycarbonates, polyether-carbonates, polyether-estercarbonates, polkyhydroxyalkanoates, polyamides and polymers that aresynthesized from one or more monomers from the group of l-lactide,dl-lactide, glycolide, ε-caprolactone, trimethylene carbonate,morpholine-dione, p-dioxanone and 1,5-dioxapan-2-one.

In one aspect, the porogen can be leeched out of the hydrogel during thedevice manufacturing process. This can be accomplished by incubating theporogen containing hydrogen in a solvent in which the porogen willdissolve. The solvent is preferably a water miscible solvent. In anotheraspect, the porogen can remain in the device throughout themanufacturing process and will degrade and leech out once the hydrogelplug is inserted into the patient.

In one aspect the plug comprises a degradable polymer. Degradablepolymers that can be used in the plug include but are not limited todegradable polyesters, polyanhydrides, polyurethanes, polyether-esters,polycarbonates, polyether-carbonates, polyether-ester carbonates,polkyhydroxyalkanoates, polyamides and polymers that are synthesizedfrom one or more monomers from the group of l-lactide, dl-lactide,glycolide, ε-caprolactone, trimethylene carbonate, morpholine-dione,p-dioxanone and 1,5-dioxapan-2-one.

The plug can comprise a monofilament structure, a multifilamentstructure, or a braided structure. In one aspect, the plug can beprepared by taking particles or chopped fibers of the degradable polymerand compression mold them into a shape. Heat can be used to thermallyfuse the particulates together such that a porous structure is obtained.In one aspect, the shape can be in the form of a rod. The porous rod canthen be cut to a predetermined length.

In one aspect, the plug can be made from an electrospun degradablepolymer. In one aspect, the plug is made from a thin film of electrospunderivatized polyhydric polymer and/or compositions. The plug can be cutdirectly from a sheet of the electrospun composition. In one aspect, theplug can be prepared by rolling an electrospun film into a roll. Theelectrospun plug or the rolled rod shaped structure can be coated with asecond degradable polymer such that the rolled configuration isretained. In one aspect, the polymer used to prepare the rolledstructure has a degradation time that is longer than the polymer used tocoat the rolled structure. This can allow the plug to be more rigidwhich makes handling easier during manufacturing but upon delivery tothe desired site, the faster degrading material will start degrading andfacilitate tissue ingrowth while the first longer lasting polymerprovides a scaffold for the ingrowing tissue.

In another aspect, the electrospun plug can be coated or dipped into asolution of a water-soluble polymer. The plug is then dried at ambientpressure or at reduced pressure. The plug may also be dried bylyophilization. The presence of the water soluble polymer can make theelectrospun composition more rigid and thus easier to handle duringmanufacturing and delivery to the intended site. Once positioned at theintended site, the polymer will start to dissolve and leech out of theelectrospun composition. The tissue from the mechanically damagedfallopian tube can then grow into the electrospun composition. Theelectrospun composition will degrade over time leaving an occludedfallopian tube. In one aspect, the water soluble polymer can be selectedfrom the group of polyethylene oxide, polyethylene glycol, blockcopolymers of polyethylene glycol and polypropylene glycol (e.g.Pluronics F126 and Pluronics F68, Sigma-Aldrich Corp., St. Louis, Mo.,USA), dextran, hyaluronic acid, or a hyaluronic acid derivative of thisdisclosure.

The degradable polymer used to form the plug can further comprise aporogen. The porogen can comprises an inorganic salt, an organic smallmolecule or a polymer. The porogen is selected such that it is solublein a solvent in which the biodegradable polymer used to prepare the plughas limited solubility.

Inorganic salts that can be used as porogens include but not limited tosodium salts, potassium salts, calcium salts, magnesium salts, aluminumsalts, copper salts, barium salts, iron salts. Examples of these saltsinclude but are not limited to sodium chloride, sodium bromide, sodiumiodide, sodium sulfate, sodium phosphate, sodium hydrogen phosphate, orcombinations thereof.

A porous plug can be prepared by 3D-printing the plug. A degradablepolymer can be used to 3D print the plug. In one aspect, the degradablepolymer that can be used in the plug include but are not limited todegradable polyesters, polyanhydrides, polyurethanes, polyether-esters,polycarbonates, polyether-carbonates, polyether-ester carbonates,polkyhydroxyalkanoates, polyamides and polymers that are synthesizedfrom one or more monomers from the group of l-lactide, dl-lactide,glycolide, ε-caprolactone, trimethylene carbonate, morpholine-dione,p-dioxanone and 1,5-dioxapan-2-one.

The plug can comprise position retaining features. These features caninclude non-symmetrical shapes, barbs, ridges, pores, slits, slots, or acombination thereof. The barbs can be unidirectional in that they allpoint in the same direction or the barbs could point in two or moredifferent directions. The barbs could be uniformly spaced on the plug orthey could be present in only specific portions of the plug.

In one aspect, plug can be dipped into a solution of the derivatizedpolyhydric polymers of the disclosure. The solution can then beactivated to allow the solution to crosslink such that the pores of theplug comprise the crosslinked derivatized polyhydric polymer. Thecrosslinking process can be activated by adjusting pH of the solution,addition of a crosslinking agent, elevation of temperature, addition ofan initiator or a combination of one or more of these.

In one aspect, the derivatized polyhydric polymer compositions of thedisclosure can be used as a scaffold to allow the ingrowth of tissue orbone. In one aspect, derivatized polyhydric polymers of this disclosurecan be prepared as a crosslinked matrix that is then lyophilized. Thelyophilized derivatized polyhydric polymer composition can then berehydrated in the presence of cells such that the hydrated matrix actsas a scaffold that allows the growth of the cells on and into thescaffold. In another aspect, the derivatized polyhydric polymers of thisdisclosure that have residual vinyl sulfone groups, can be electrospunto form a porous matrix. The electospun fibers can then be crosslinkedusing heat, ultraviolet, e-beam or gamma radiation. In another aspect,the derivatized polyhydric polymer of the disclosure that containsresidual vinyl sulfone groups can further comprise a photocrosslinker. Asolution of this composition can be electrospun and then the electrospunmatrix can be subjected to ultraviolet radiation such that thephotocrosslinker results in crosslinking of the derivatized polyhydricpolymer. The resultant matrix can be rehydrated in the presence of cellssuch that it acts as a scaffold for tissue growth. In another aspect,carboxylic acid containing derivatized polyhydric polymers of thisdisclosure can be electospun into a matrix by mixing a solution of thederivatized polyhydric polymer of this disclosure with a solution of amultivalent cation just prior to electrospinning. In one aspect, asolution of a carboxylic acid containing composition of this disclosurecould be placed in one syringe and a solution of a multivalent cation ora cationic polymer can be placed in another syringe. The syringes can beconnected via a y-connector and a needle can be connected to final armof the y-connector. They two solutions can then be pumped through theneedle and this mixture can be electrospun onto a surface such that thederivatized polyhydric polymer of the disclosure is ionicallycrosslinked. Multivalent cations can include calcium magnesium, ferricions, ferrous ions, aluminum and chromium.

Cationic polymers that can be used include but are not limited tochitosan and derivatives thereof, polyvinyl pyrollidone, peptidescontaining more than one lysine group and polyethyleneimine.

In another aspect, a solution of a derivatized polyhydric polymercompositions of this disclosure can be used to coat a degradable ornon-degradable scaffold matrix. In one aspect, a derivatized polyhydricpolymer of this disclosure that has been modified with alkyl or arylgroups can be used to coat a scaffold for tissue growth. The alkyl oraryl groups will interact with the scaffold through hydrophobic bondwhile the hydrophilic portion of the derivatized polyhydric polymer willallow for cell growth on the coated scaffold surface. In another aspect,the derivatized polyhydric polymers of this disclosure that haveresidual vinyl sulfone groups, can be coated onto the scaffold. Thecoated scaffold can be subjected to heat which will result in thederivatized polyhydric polymer transforming into a crosslinkedderivatized polyhydric polymer.

In another aspect, the derivatized polyhydric polymers and/orcompositions of the disclosure can comprise a sulfonate group. Inanother aspect, the derivatized polyhydric polymers and/or compositionsof the disclosure can comprise both hydrophobic groups and sulfonategroups. The hydrophobic groups can be alkyl or aromatic based.

In another aspect, tissue scaffold support structure can be 3D printedor electrospun using a degradable polymer. The degradable polymer thatcan be used can include but not limited to degradable polyesters,polyanhydrides, polyurethanes, polyether-esters, polycarbonates,polyether-carbonates, polyether-ester carbonates,polkyhydroxyalkanoates, polyamides and polymers that are synthesizedfrom one or more monomers from the group of l-lactide, dl-lactide,glycolide, ε-caprolactone, trimethylene carbonate, morpholine-dione,p-dioxanone and 1,5-dioxapan-2-one.

In another aspect for the electrospun scaffold support structure, asingle or multiple polymer solutions can be prepared. The polymers usedcan be biodegradable polymers then include but are not limited topolyester, polyanhydride, polyorthoester, polycarbonate,poly-ester-co-carbonate), polyhydroxybutyrates or combinations thereof.Biodegradable polymers can include polylactice-co-glycolide copolymers,polydioxanone, polylactide-trimethylene carbonate copolymers as well ascopolymers that comprise repeat units derived from at least one of thefollowing monomers: l-lactide, dl-lactide, glycolide, trimethylenecarbonate, epsilon-caprolactone, p-dioxanone and a morpholinedione

The solvents used can be an organic solvent, water or a combinationthereof. For example, HFIP, DMSO, NMP, Chloroform, acetic acid, ethanol,dimethylformamide (DMF) solvents or mixtures of solvents can be used.Solutions with a concentration of 0.5 to 25% (w/v) can be prepared. Thesolution that is to be electrospun can be placed in a syringe with aneedle. The syringe is then placed in a syringe pump. The needle canhave a blunt end and an inner diameter in the range of 0.25 to 2.5 mm.The needle and collection plate are attached to a high voltage supply.In some applications, more than one needle can be used to prepare asingle sheet. The needles can be arranged such that the same polymersolution flows through all the needles, different solutions flow throughdifferent needles or a combination thereof. The needles can be arrangedsuch that adjacent needles allow different polymer solutions to flowthrough them. This alternation pattern can be repeated. A voltage isthen applied to the system. The applied voltage can be in the 10 kV to45 kV. The syringe pump can extrude the solution. The flow rate of thesyringe pump can be in the range of 0.0001 uL/min to 423 mL/min. Thecollector plate can be static, rotating or moving in a specific lineardirection to give the fibers some directional orientation. The shape ofthe collector plate can be varied with the collector plate having butnot limited to the following shapes: a flat surface, a textured surface,a curved surface, a square rod, a rectangular rod, a round mandrel, anoval mandrel, a semi-circular mandrel or a combination of these shapes.The distance of the needle tip to the collector plate can be altered.The distance of the needle tip to the collector plate can be in the 2-50cm range. The collection plate can also be submerged in or sprayed witha solvent that assists in the precipitation of the newly spun fibers.For example, an ethanol bath may be used during the electrospinning ofhyaluronic acid based derivatized polyhydric polymers of thisdisclosure. The derivatized polyhydric polymer of the disclosure can beincorporated through a solution coating or submersion of an electrospunmatrix.

In one aspect the polymer composition used to 3D print or electrospinthe scaffold can further comprise an inorganic filler or a combinationof inorganic fillers. In one aspect the inorganic filler can be selectedfrom the group calcium carbonate, calcium phosphate, tricalciumphosphate, hydroxyapatite, bioglass, or a combination thereof.

In one aspect, 3D-printed or electrospun scaffold can be coated with asolution of the derivatized polyhydric polymers of the disclosure. Thisderivatized polyhydric polymer can be coated onto the scaffold through adip coating or spray coating process. In another aspect, the derivatizedpolyhydric polymer can be dispersed into the scaffold throughcompressive application. In another aspect, the derivatized polyhydricpolymer can be dispersed into the scaffold through submersion insolution which may or may not include sonication to aid in dispersion.In another aspect, the coated scaffold can be dried. The drying processcan include drying at elevated temperature, drying at reduced pressureor lyophilization. In another aspect, the solution of the derivatizedpolyhydric polymer compositions of the disclosure can further comprise abiologically active agent.

In another aspect, scaffold can be dipped into a solution of thederivatized polyhydric polymers of the disclosure. The solution can thenbe activated to allow the solution to crosslink such that the pores ofthe scaffold comprise the crosslinked derivatized polyhydric polymer.The crosslinking process can be activated by adjusting pH of thesolution, addition of a crosslinked, elevation of temperature, additionof an initiator or a combination of one or more of these.

In another aspect, scaffold can be dipped into a solution of thederivatized polyhydric polymers of the disclosure and allowed to dry orbe lyophilized. The derivatized polyhydric polymers within the substratecan then be dipped into a crosslinking solution to allow the solution tocrosslink such that the pores of the scaffold comprise the crosslinkedderivatized polyhydric polymer. The crosslinking process can beactivated by adjusting pH of the solution, addition of a crosslinked,elevation of temperature, addition of an initiator or a combination ofone or more of these.

In another aspect, scaffold can be dipped into a solution of thederivatized polyhydric polymers of the disclosure and a crosslinkingagent. The rate of the crosslinking reaction can be controlled such thatthe scaffold can be coated with the derivatized polyhydric polymerand/or composition prior to complete crosslinking of the derivatizedpolyhydric polymer. In one aspect a biologically active agent can beincorporated into the derivatized polyhydric polymer and/or compositionsbefore or immediately following the initiation of the crosslinkingreaction. The scaffold can then be coated with this composition and onceapplied to the scaffold, the crosslinking reaction is completed suchthat the device comprises the crosslinked derivatized polyhydric polymerwith the biologically active agent essentially encapsulated by thecrosslinked derivatized polyhydric polymer composition.

In another aspect, the derivatized polyhydric polymers and/orcompositions of this disclosure are used to prepare a scaffold or tocoat the scaffold can comprise a biologically active agent. In oneaspect, the biologically active agent can enhance cell growth. In oneaspect, the biologically active agent can be one or more growth factorsor peptides that enhance cell growth and cell adhesion. In anotheraspect, the derivatized polyhydric polymers and/or compositions of thisdisclosure used to prepare a scaffold or to coat the scaffold canfurther comprise an excipient. In one aspect, the derivatized polyhydricpolymer compositions of the disclosure can comprise one or moreextracellular matrix components. The extracellular matrix component caninclude but are not limited to heparan sulfate, chondroitin sulfate,keratin sulfate, hyaluronic acid, collagen, elastin, fibronectin, andlaminin.

In one aspect, the cells that can be added to the scaffolds that containthe derivatized polyhydric polymer compositions of this disclosureinclude embryonic stem cells, mesenchymal stem cells, adipose-derivedstem cell, endothelial stem cells, dental pulp stem cells, tumor cells,chondrocytes, osteoblasts, dermal fibroblasts, protomyofibroblasts,myofibroblasts, hepatocytes, smooth muscle cells, endothelial cells,epithelial cells, adipose tissue, adipose cells and cardiac cells

In one aspect, the derivatized polyhydric polymers and compositions ofthe present disclosure comprise free vinyl sulfone functional groups andcan be used to 3D print structures. The derivatized polyhydric polymerscan be prepared as solutions with viscosities that allow them to be 3dprinted. In one aspect, a solution of the derivatized polyhydric polymerwith residual vinyl sulfone groups can be prepared. A second solutioncontaining a derivatized polyhydric polymer with at least two free thiolgroups can be prepared. In one aspect, the first and second solution canbe mixed together. Just prior to printing, the pH of the mixture can beadjusted to a pH of greater than 8, preferably greater than 9, such thatthe mixture can be printed and then cure following printing. In oneaspect, the pH can be adjusted by mixing the mixture with a buffersolution that has a pH of greater than 8. The mixing takes place justprior to the print head ensuring that the mixture does not gel up in theprint head and thus clot the printer, In another aspect, the solution ofthe derivatized polyhydric polymer that comprises the residual vinylsulfone functional groups can has its pH adjusted to a pH of greaterthan 8 by mixing it with a buffer solution. This solution can then bemixed with solution 2 just prior to the print head such that the mixtureis printed and then allowed to complete gelation once printed.

The viscosity of the mixture can be used to control the retention of theprinted structure until gelation is completed. In another aspect, athermogelling material can be added to either the first, second orbuffer solution. Thus, the mixture can be printed and then thetemperature of the printed environment can be different from thesolution prior to printing such that following the printing process theprinted solution undergoes thermal gelation to preserve the initialprinted structure while the crosslinking process is moving towardscompletion.

Thermogelling materials can include but are not limited topolyethylene-block-polypropylene co polymers such as Pluronics F127 orF68 (Sigma-Aldrich Corp., St. Louis, Mo., USA) or polyester-polyethyleneglycol block co polymers. The polyester-polyethylene glycol copolymerscan include deblock and triblock copolymers. The polyester component arepolymers that are synthesized from at least one of the monomers from thegroup of l-lactide, dl-lactide, glycolide, ε-caprolactone,morpholine-dione, p-dioxanone and 1,5-dioxapan-2-one. In another aspect,a thermogelling polymer that comprises trimethylene carbonate can beused.

Following the completion of the gelation process, the printed constructcan be rinsed to neutralize the pH of the printed gel. In another aspectthe printed structure can be dried such that the residual water contentis less than 10%. In another aspect, the printed structure can belyophilized.

The printed structure can be used as a tissue scaffold, for woundhealing applications, for occlusion of a lumen, a biopsy site or aneedle tract.

For procedures such as neuroendoscopy, intracranial decompression, andtreatment of chronic subdural hematoma, holes are often drilled into theskull. These are often referred to as burr holes. In many instances,these burr holes are left untreated following the surgical procedure andthe scalp is replaced directly over these holes. This can lead to scalpdepressions at the burr hole. These scalp depressions can lackmechanical strength. In order to prevent this, a burr hole plug can beinserted into the burr hole such that it can facilitate and support boneregrowth. Autologous bone can be used to fill the burr holes but thisrequires harvesting of the bone. Synthetic materials can be used as burrhole plugs. A degradable burr hole plug that degrades while facilitatingbone ingrowth will allow the healing of the burr hole without leavingresidual material. A polycaprolactone (PCL) burr hole plug has beencommercialized. The challenge with PCL is that it is slow degrading andthe interface between the polymer and the in-growing tissue is usuallynot the best due to the hydrophobicity of the polymer.

The derivatized polyhydric polymers and compositions thereof of thedisclosure can be made into a burr hole plug. A solution of aderivatized polyhydric polymer can be placed in the mold and then thederivatized polyhydric polymer composition can be lyophilized to producea porous structure that can be inserted into the burr hole. In anotheraspect, the derivatized polyhydric polymer compositions of thedisclosure can be electrospun and then cut to form a plug that can beinserted into the burr hole. In another aspect, a solution of thederivatized polyhydric polymer of the disclosure can be placed in a moldand the solution can be crosslinked. The crosslinked plug can be useddirectly. In another aspect, the crosslinked derivatized polyhydricpolymer compositions can be lyophilized to yield a porous crosslinkedstructure that can be used as a burr hole plug.

In another aspect, a burr hole plug can be 3D printed or electrospunusing a degradable polymer. The degradable polymer that can be used caninclude but not limited to degradable polyesters, polyanhydrides,polyurethanes, polyether-esters, polycarbonates, polyether-carbonates,polyether-ester carbonates, polkyhydroxyalkanoates, polyamides andpolymers that are synthesized from one or more monomers from the groupof l-lactide, dl-lactide, glycolide, ε-caprolactone, trimethylenecarbonate, morpholine-dione, p-dioxanone and 1,5-dioxapan-2-one.

In one aspect the polymer used to 3D print or electrospin the burr holeplug can further comprise an inorganic filler or a combination ofinorganic fillers. In one aspect the inorganic filler can be selectedfrom the group calcium carbonate, calcium phosphate, tricalciumphosphate and hydroxyapatite.

In one aspect, the 3d-printed or electrospun burr plug can furthercomprise an extracellular matrix material. In one aspect, theextracellular matrix material can be selected from the group collagen,hyaluronic acid, chondroitin sulfate, heparan sulfate, keratin sulfate,elastin, fibronectin and laminin.

In one aspect, 3D-printed or electrospun plug can be coated with asolution of the derivatized polyhydric polymers of the disclosure. Thisderivatized polyhydric polymer composition can be coated onto the plugthrough a dip coating or spray coating process. In another aspect, thecoated plug can be dried. The drying process can include drying atelevated temperature, drying at reduced pressure or lyophilization.

In another aspect, polymeric degradable plug can be dipped into asolution of the derivatized polyhydric polymers of the disclosure. Thesolution can then be activated to allow the solution to crosslink suchthat the pores of the plug comprise the crosslinked derivatizedpolyhydric polymer composition. The crosslinking process can beactivated by adjusting pH of the solution, addition of a crosslinker,elevation of temperature, addition of an initiator or a combination ofone or more of these.

In another aspect, polymeric degradable plug can be dipped into asolution of the derivatized polyhydric polymers of the disclosure thatcontain residual vinyl sulfone groups. The coateddevice can be dried atelevated temperatures to remove the solvent and to allow crosslinking ofthe coating such that the pores of the plug comprise the crosslinkedderivatized polyhydric polymer composition.

In one aspect, the crosslinked forms of the derivatized polyhydricpolymers and/or compositions of this disclosure can be used to formnerve guides. Optionally, the nerve guides can be prepared bylyophilization. In one aspect, collagen, gelatin, chitosan heparansulfate or a combination of these can be further added to thederivatized polyhydric polymers and/or compositions of the disclosure toform the nerve guides. In another aspect, Schwann cells can beincorporated into the derivatized polyhydric polymer compositions duringthe formation of the nerve guide.

In one aspect, the derivatized polyhydric polymers if this disclosurecan be prepared as a solution that has a viscosity of greater than 50cP. In one aspect, this solution can be applied to tissue to reduce thecoefficient of friction with the tissue surface. In one aspect, thederivatized polyhydric polymer composition can be used as a vaginallubricant. In another aspect, the solution can be applied to a devicethat is to be inserted into an opening, orifice or cavity such that thesolution act to lubricate the passage of the device through the opening,orifice or cavity. In one aspect the device could be an endoscope.

In one aspect, the derivatized polyhydric polymer and compositionsthereof of this disclosure can be used to coat a medical device. Medicaldevices that can be coated include but are not limited to a catheter, aneedle, a biopsy needle, a tissue marker, a guide wire, and endoluminalsheath, a suture, a braid, a trocar, a hernia mesh, a surgical mesh, acontact lens, an intra-ocular lens, a stent (for example vascular stent,esophageal stent, biliary stent coronary stent, renal stent, peripheralvascular stent), a nasal splint, a vascular graft, a stent-graft,aneurysm coils, introducer sheaths, balloon catheters, vascular closuredevices, inferior vena cava filter, and Hydrocephalic shunts.

In one aspect, the derivatized polyhydric polymer of the disclosure canbe prepared as a solution which can then be applied by spray coating ordip coating. The solvent can then be removed to leave a coating of thederivatized polyhydric polymer composition of the disclosure on thedevice surface. In one aspect, the solution can be an aqueous solution.In another aspect, the solution can comprise an organic solvent. Inanother aspect, the solution can comprise water and a water-miscibleorganic solvent. In one aspect the derivatized polyhydric polymer of thedisclosure can be functionalized with aliphatic or aromatic groups suchthat there is a hydrophobic interaction with these groups and the devicesurface. In one aspect, the derivatized polyhydric polymer of thisdisclosure that has residual vinyl sulfone groups can be coated onto amedical device by dip coating or spray coating. The coating is dried.The coating can be exposed to heat, gamma, e-beam or ultravioletradiation to crosslink the derivatized polyhydric polymer. In anotheraspect, the coating can further comprise a biologically active agent. Inanother aspect, the coating when hydrated, increase the lubricity of thecoated device. The increased lubricity of the coated device can bemeasure by a decrease of the water contact angle by at least 20′. Inanother aspect, the increased lubricity can be measured as a decrease inthe friction coefficient by at least 20%. In another aspect, the devicecan be partially coated with some part of the device remaining uncoated.In another aspect, the device can be precoated with binding polymercoating that enhances the binding of the coating derivatized polyhydricpolymer composition of this disclosure. In another aspect, the coatingcan further comprise heparin, to give the coating anti-thromboticproperties.

A process for making a derivative polymer of a polyhydric polymer,comprising:

a) reacting hydroxyl groups of a polyhydric polymer, with divinylsulfone (DVS) to provide a first polyhydric derivative; and

b) reacting the first polyhydric polymer derivative with a nucleophileof a formula X′—R¹—Y, or X′—R²—Y₇ or both to provide a second polyhydricpolymer derivative; wherein R¹ and R² are different, and each is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, X′comprises a nucleophilic group of SH or NH₂, and Y is the same ordifferent, and Y is one or more of H, a carboxylic acid group or a saltor ester thereof, a hydroxyl group, a sulfonic acid group or a saltthereof, or an amine group. This process, wherein the polyhydric polymeris hyaluronic acid (HA). This process may further further comprise stepc) derivatizing the second polyhydric polymer derivative by repeating,one or more times, step a) or step a) and step b).

The following Examples are offered by way of illustration and not by wayof limitation. In the Examples, DI stands for distilled water, PEGstands for polyethylene glycol and IV stands for intrinsic viscosity.

EXAMPLES Example 1 DVS Modified HA (DVS2)

2.5 g sodium hyaluronate (900 KDa) was added to a glass 4 L reactionkettle. The lid, overhead stirrer and anchor impellor were attached tothe reaction kettle. The solution was then stirred at about 200 rpm. 250g deionized water was added to the kettle. The solution was stirred forabout 18 hrs. 166.5 g of a 0.25 M NaOH solution was added to thedissolved sodium hyaluronate. The pH of the solution was measured after2 min and was found to be 12.69. A freshly prepared solution of 10.6 gdivinyl sulfone in 66 g of DI water was then rapidly added to thestirring solution. After 75 seconds, 50 g of a 1M HCl solution was addedto the reaction mixture. 1 M NaOH was then added dropwise until thesolution pH was between 5 and 7. 6 g NaCl was then added to thesolution. Once the NaCl had dissolved, 1.25 L acetone was slowly addedover a period of 20 minutes. The suspension was stirred for about 3hours. 200 mL denatured ethanol was added and the solution was stirredfor about 30 minutes. The precipitate was filtered under vacuum using asintered glass funnel through a 0.22 μm PTFE filter membrane. Once allthe solution had been filtered, the vacuum was disconnected and 100 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temp in a vacuum oven.

Approx. 10-20 mg of the dried sample was added to a vial. D₂O was addedto the sample to make the final concentration of the solution about 6mg/mL. The sample was shaken on an orbital shaker until dissolved. Oncedissolved, the sample was transferred into a NMR tube and the ¹H-NMRspectrum of the sample was recorded on a NMR spectrometer. The spectrumwas printed out with the specific peaks in the 6.3-6.5 ppm (2 peaks fromthe 2 CH₂=protons from the vinyl sulfone residue), the 6.8-7.0 ppm (CHpeak of vinyl group) and 1.8-2.5 ppm (singlet from the 3 CH₃ protonsfrom the N-acetyl group of the HA) regions being integrated. The percentmodification is calculated on molar ratio of the vinyl CH protons (6.8-7ppm) to the acetamide (1.8-2.5 ppm) protons. The percent substitutionwas found to be about 8.9%. The ¹H-NMR spectrum of the sample is shownin FIG. 1.

Example 2 DVS Modified HA (DVS13)

3.5 g sodium hyaluronate (approx. 800 kDa; 1.4 m3/Kg IV) was added to a4 L glass reaction kettle. The lid, overhead stirrer and anchor impellorwere attached to the reaction kettle. 350 g deionized water was added tothe kettle. The solution was then stirred at about 300 rpm. The solutionwas stirred for about 18 hrs. The stirring speed was then increased to750 rpm and about 233 g of a 0.25 M NaOH solution was added to thedissolved sodium hyaluronate. The pH of the solution was measured after2min and was found to be 12.95. A freshly prepared solution of 15.5 gdivinyl sulfone in 92.4 g of DI water was then rapidly added to thestirring solution. After 4.5 minutes, 63 g of a 1 M HCl solution wasadded to the reaction mixture. 1 M NaOH was then added dropwise untilthe solution pH was between 5 and 7. 8.4 g NaCl was then added to thesolution. Once the NaCl had dissolved, 1.5 L acetone was slowly addedover a period of 30 minutes. The suspension was stirred for about 3hours. 300 mL denatured ethanol was added and the solution was stirredfor about 30 minutes. The precipitate was filtered under vacuum using asintered glass funnel through a 0.22 μm PTFE filter membrane. Once allthe solution had been filtered, the vacuum was disconnected and 150 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temp in a vacuum oven. Thepercent substitution, as determined by the procedure described inExample 1, was found to be about 25%.

Example 3 DVS Modified HA (DVS14)

The reaction as described in Example 2 was performed using a reactiontime of 6 minutes. The percent substitution, as determined according tothe procedure described in Example 1, was found to be about 31%.

Example 4 HA-DVS Reaction with 3-Mercaptopropionic Acid (HA-DVS2-MPA)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 1) wasadded to 50 g DI water in a 250 mL round bottom flask. The solution wasstirred overnight until the material had dissolved. The flask was thenpurged with nitrogen. 0.022 g 3-mercaptopropionic acid (MPA) was addedto the solution. After the MPA had dissolved, the pH was adjusted toabout 9 using 0.25 M NaOH. The solution was stirred for 4 hours afterwhich the pH was adjusted to about 7 using 0.25 M HCl. 1.25 g NaCl wasadded to the reaction solution. The solution was stirred until the NaClhad dissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. 25 mL ethanol was added andthe resultant mixture was stirred for 15 minutes. The precipitate wasisolated using vacuum filtration. The precipitate was washed 4 timeswith 25 mL ethanol in such a manner that the filter funnel did not rundry. The precipitate was dried under vacuum at room temperature. Asample of the material was dissolved in D₂O and the ¹H-NMR spectrum wasmeasured. The presence of MPA substitution was evidenced by peaks at2.3-2.4 ppm (triplet) and 2.6-2.8 ppm (triplet). The MPA substitution,as calculated from the integrals at 2.3-2.4 ppm (MPA-H₂) and 1.7-2 ppm(HA-acetamide), was 7.6%.

Example 5 HA-DVS Reaction with 1-octanethiol (HA-DVS2-oct)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 1) wasadded to 50 g DI water in a 250 mL round bottom flask. The solution wasstirred for about 4 hours at room temperature. About 15.8 g denaturedethanol was added and the mixture was stirred for about 18 hrs at whichpoint the material had dissolved. The flask was then purged withnitrogen. 0.023 g 1-octanethiol in 7.9 g ethanol was then added to thesolution of derivatized HA. The pH of the reaction mixture was adjustedto about 9 using 0.25 M NaOH. The solution was stirred for 4 hours afterwhich the pH was adjusted to about 7 using 0.25 M HCl. 0.5 g NaCl wasadded to the reaction solution. The solution was stirred until the NaClhad dissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. The precipitate was isolatedusing vacuum filtration. The precipitate was washed 4 times with 25 mLethanol in such a manner that the filter funnel did not run dry. Theprecipitate was dried under vacuum at room temperature. A sample of thematerial was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thepresence of octanethiol substitution was evidenced by peaks at 0.8-0-9ppm (—CH₃), 1.2-1.6 ppm (—CH₂—), 2.6-2.7 ppm (—CH₂—S—) and 2.9-3.0 ppm(—S—CH2-). The octanethiol molar substitution, as calculated from theintegrals at 2.6-2.7 ppm. (Oct-CH₂—S—) and 1.7-2 ppm (HA-acetamide), was5.4%.

Example 6 HA-DVS Reaction with 1-octanethiol (HA-DVS2-oct-DMF)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 1) wasadded to 50 g DI water in a 250 mL round bottom flask. The solution wasstirred for about 4 hours at room temperature. About 18.88 gdimethylformamide (DMF) was added and the mixture was stirred for about18 hrs at which point the material had dissolved. The flask was thenpurged with nitrogen. 0.029 g 1-octanethiol in 9.4 g DMF was then addedto the derivatized HA solution. The pH of the reaction mixture wasadjusted to about 9 using 0.25 M NaOH. The solution was stirred for 4hours after which the pH was adjusted to about 7 using 0.25 M HCl. About0.25 g NaCl was added to the reaction solution. The solution was stirreduntil the NaCl had dissolved. 150 mL cold acetone was slowly added tothe solution. The reaction mixture was stirred for 1.5 hours. 25 mLethanol was added and the resultant mixture was stirred for 15 minutes.The precipitate was isolated using vacuum filtration. The precipitatewas washed 4 times with 25 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D₂O and the¹H-NMR spectrum was measured. The octanethiol molar substitution, ascalculated from the integrals at 2.4-2.5 ppm (Oct-CH₂—S—) and 1.7-2 ppm(HA-acetamide), was 5.5%.

Example 7 HA-DVS Reaction with 1-dodecanethiol (HA-DVS2-dod)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 1) wasadded to 50 g DI water in a 250 mL round bottom flask. The solution wasstirred for about 4 hours at room temperature. About 15.8 g denaturedethanol was added and the mixture was stirred for about 18 hrs at whichpoint the material had dissolved. The flask was then purged withnitrogen. 0.04 g 1-dodecanethiol in 7.9 g ethanol was then added to thesolution of derivatized HA. The pH of the reaction mixture was adjustedto about 9 using 0.25 M NaOH. The solution was stirred for 4 hours afterwhich the pH was adjusted to about 7 using 0.25 M HCl. 0.25 g NaCl wasadded to the reaction solution. The solution was stirred until the NaClhad dissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. The precipitate was isolatedusing vacuum filtration. The precipitate was washed 4 times with 25 mLethanol in such a manner that the filter funnel did not run dry. Theprecipitate was dried under vacuum at room temperature. A sample of thematerial was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thepresence of octanethiol substitution was evidenced by peaks at 0.8-0-9ppm (CH₃—), 1.2-1.6 ppm (—CH₂—), 2.6-2.7 ppm (—CH₂—S—) and 2.9-3.0 ppm(—S—CH₂—). The octanethiol molar substitution, as calculated from theintegrals at 2.6-2.7 ppm (Oct-CH₂—S—) and 1.7-2 ppm (HA-acetamide), was5.2%.

Example 8 DVS Modified HA—Reaction 2 (DVS3)

3.5 g sodium hyaluronate (approx. 900 kDa, 17 dL/g) was added to a glass4 L reaction kettle. The lid, overhead stirrer and anchor impellor wereattached to the reaction kettle. 350 g deionized water was added to thekettle. The solution was stirred at about 200 rpm for about 18 hrs. 233g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be 12.6. A freshly prepared solution of 14.8 g divinyl sulfonein 92.4 g of DI water was then rapidly added to the stirring solution.After 1.25 minutes, 70 g of 1M HCl was added to the reaction mixture.Either 1 M NaOH or 1M HCl was then added dropwise as needed until thesolution pH was between 5 and 7. About 6 g NaCl was then added to thesolution. Once the NaCl had dissolved, 1.25 L acetone was slowly addedover a period of 20 minutes. The suspension was stirred for about 3hours. 200 mL ethanol was added and the solution was stirred for about30 minutes. The precipitate was filtered under vacuum using a sinteredglass funnel through a 0.22 μm PTFE filter membrane. 100 mL ethanol wasused to rinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times. The productwas dried under vacuum at room temp in a vacuum oven. The percentsubstitution, determined as described in Example 1, was found to be8.6%.

Example 9 DVS Modified HA—(DVS5—800 kDa)

3.5 g sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) was added to aglass 4 L reaction kettle. The lid, overhead stirrer and anchor impellorwere attached to the reaction kettle. 350 g deionized water was added tothe kettle. The solution was stirred at about 200 rpm for about 18 hrs.233 g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be 12.85. A freshly prepared solution of 14.8 g divinyl sulfonein 92.4 g of DI water was then rapidly added to the stirring solution.After 75 seconds, 63 g of a 1M HCl solution was added to the reactionmixture. Either 1M NaOH or 1M HCl was then added dropwise as neededuntil the solution pH was between 5 and 7. 8.4 g NaCl was then added tothe solution. Once the NaCl had dissolved, 1. 5 L acetone was slowlyadded over a period of 30 minutes. The suspension was stirred for about3 hours. 200 mL ethanol (Ethanol, Alcohol Reagent, Denatured anhydrous94-96%) was added and the solution was stirred for about 30 minutes. Theprecipitate was filtered under vacuum using a sintered glass funnelthrough a 0.22 μm PTFE filter membrane. 150 mL ethanol was used to rinsethe precipitate. The ethanol was then removed by vacuum filtration. Thisprocess was repeated an additional 3 times. The product was dried undervacuum at room temp in a vacuum oven. The percent substitution,determined as described in Example 1, was found to be about 8.9%.

Example 10 HA-DVS Reaction with 1-pentanethiol (HA-DVS5-pent2)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 9) wasadded to 27.5 g DI water in a 250 mL round bottom flask. The solutionwas stirred for about 4 hours at room temperature. About 16 g denaturedethanol was added and the mixture was stirred for about 18 hrs at whichpoint the material had dissolved. The flask was then purged withnitrogen. 0.042 g 1-pentanethiol in about 1.8 g ethanol was then addedto the solution of derivatized HA. The pH of the reaction mixture wasadjusted to about 9 using 0.25 M NaOH. The solution was stirred for 4hours after which the pH was adjusted to about 7 using 0.25 M HCl. About0.5 g NaCl was added to the reaction solution. The solution was stirreduntil the NaCl had dissolved. 150 mL cold acetone was slowly added tothe solution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. The precipitate waswashed 4 times with 25 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample was dissolved in D₂O and the ¹H-NMR spectrum wasmeasured. The presence of pentanethiol substitution was evidenced bypeaks at 0.6-0-8 ppm (CH₃—), 1.2-1.6 ppm (—CH₂—), 2.4-2.6 ppm (—CH₂—S—)and 2.7-2.9 ppm (—S—CH₂—). The pentanethiol molar substitution, ascalculated from the integrals at 2.3-2.7 ppm (pent-CH2—S—) and 1.7-2 ppm(HA-acetamide), was 7.3%.

Example 11 HA-DVS Reaction with 1-decanethiol (HA-DVSS-dec)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 9) wasadded to 20 DI water in a 250 mL round bottom flask. The solution wasstirred for about 4 hours at room temperature. 19.7 g denatured ethanolwas added and the mixture was stirred for about 18 hrs at which pointthe material had dissolved. The flask was then purged with nitrogen.0.035 g 1-decanethiol in 4 g ethanol was then added to the derivatizedHA solution. The pH of the reaction mixture was adjusted to about 9using 0.25 M NaOH. The solution was stirred for 4 hours after which thepH was adjusted to about 7 using 0.25 M HCl. 0.25 g NaCl was added tothe reaction solution. The solution was stirred until the NaCl haddissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. The precipitate was isolatedusing vacuum filtration. The precipitate was washed 4 times with 25 mLethanol in such a manner that the filter funnel did not run dry. Theprecipitate was dried under vacuum at room temperature. A sample of thematerial was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thepresence of decanethiol substitution was evidenced by peaks at 0.6-0-8ppm (CH₃—), 1.1-1.6 ppm (—CH₂—), 2.4-2.6 ppm (—CH₂—S—) and 2.7-2.9 ppm(—S—CH2-). The decanethiol molar substitution, as calculated from theintegrals at 2.3-2.7 ppm (pent-CH₂—S—) and 1.7-2 ppm (HA-acetamide), was6.5%.

Example 12 HA-DVS Reaction with 3-mercapto-1-propanesulfonate(HA-DVSS-SMPS)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 9) wasadded to 50 g DI water in a 250 mL round bottom flask. The solution wasstirred overnight until the material had dissolved. The flask was thenpurged with nitrogen. 0.036 g sodium-3-mercapto-1-propanesulfonate(SMPS) was added to the solution. After the SMPS had dissolved, the pHwas adjusted to about 9 using 0.25 M NaOH. The solution was stirred for4 hours after which the pH was adjusted to about 7 using 0.25 M HCl.1.25 g NaCl was added to the reaction solution. The solution was stirreduntil the NaCl had dissolved. 150 mL cold acetone was slowly added tothe solution. The reaction mixture was stirred for 1.5 hours. 25 mLethanol was added and the resultant mixture was stirred for 15 minutes.The precipitate was isolated using vacuum filtration. The precipitatewas washed 4 times with 25 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D₂O and the¹H-NMR spectrum was measured. The presence of SMPS substitution wasevidenced by peaks at 2.0-2.1 ppm (—CH₂—), 2.5-2.7 ppm (—CH₂—S—) and2.8-3.0 ppm (—S—CH₂—). The SPMS substitution, as calculated from theintegrals at 2.5-2.7 ppm (SMPS—CH₂—S—) and 1.7-2 ppm (HA-acetamide), was6.4%.

Example 13 HA-DVS Reaction with cysteine (HA-DVS5-cys)

0.5 g vinyl sulfone derivatized HA (approx. 9%, as per Example 9) wasadded to 50 g DI water in a 250 mL round bottom flask. The solution wasstirred overnight until the material had dissolved. The flask was thenpurged with nitrogen. 0.024 g L-cysteine was added to the solution.After the cysteine had dissolved, the pH was adjusted to about 9 using0.25 M NaOH. The solution was stirred for 4 hours after which the pH wasadjusted to about 7 using 0.25 M HCl. 1.25 g NaCl was added to thereaction solution. The solution was stirred until the NaCl haddissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. 25 mL ethanol was added andthe resultant mixture was stirred for 15 minutes. The precipitate wasisolated using vacuum filtration. The precipitate was washed 4 timeswith 25 mL ethanol in such a manner that the filter funnel did not rundry. The precipitate was dried under vacuum at room temperature. Asample of the material was dissolved in D₂O and the ¹H-NMR spectrum wasmeasured. The presence of cysteine substitution was evidenced by peaksat 2.8-3.0 ppm (—S—CH₂—). The cysteine substitution, as calculated fromthe integrals at 2.8-3.0 ppm (—CH₂—S—) and 1.7-2 ppm (HA-acetamide), was4.9%.

Example 14 DVS Modified HA—Reaction 5 (DVS10—800 kDa)

5 g sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) was added to a glass4 L reaction kettle. The lid, overhead stirrer and anchor impellor wereattached to the reaction kettle. 500 g deionized water was added to thekettle. The solution was stirred for about at about 200 rpm for 18 hrs.333 g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be 12.93. A freshly prepared solution of 11 g divinyl sulfonein 66 g of DI water was then rapidly added to the stirring solution.After 2.5 minutes, 90 g of a 1M HCl solution was added to the reactionmixture. Either 1M NaOH or 1M HCl as needed was then added dropwiseuntil the solution pH was between 5 and 7. About 12 g NaCl was thenadded to the solution. Once the NaCl had dissolved, 1.75 L acetone wasslowly added over a period of 30 minutes. The suspension was stirred forabout 3 hours. 300 mL ethanol (Ethanol, Alcohol Reagent, denaturedanhydrous 94-96%) was added and the solution was stirred for about 30minutes. The precipitate was filtered under vacuum using a sinteredglass funnel through a 0.22 μm PTFE filter membrane. Once all thesolution had been filtered, the vacuum was disconnected and 200 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 2 times.The product was dried under vacuum at room temp in a vacuum oven. Thepercent substitution, as determined according to the procedure describedin Example 1, was found to be 8.1%.

Example 15 HA-DVS Reaction with 2-mercaptobenzoic acid (HA-DVS10-MBA)

0.5 g vinyl sulfone derivatized HA (approx. 8%, as per Example 14) wasadded to 27.5 g DI water in a 250 mL round bottom flask. The solutionwas stirred for about 4 hours at room temperature. 16 g denaturedethanol was added and the mixture was stirred for about 18 hrs at whichpoint the material had dissolved. The flask was then purged withnitrogen and then placed in a water bath (temp=30±2° C.). 0.092 g2-mercaptobenzoic acid (MBA) in 1.78 g ethanol was then added to thesolution of derivatized HA. The pH of the reaction mixture was adjustedto about 9 using 0.25 M NaOH. The solution was stirred for 4 hours afterwhich the pH was adjusted to about 7 using 0.25 M HCl. About 0.25 g NaClwas added to the reaction solution. The solution was stirred until theNaCl had dissolved. 150 mL cold acetone was slowly added to thesolution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. The precipitate waswashed 4 times with 25 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D₂O and the¹H-NMR spectrum was measured. The presence of MBA substitution wasevidenced by peaks at 7.1-7.5 ppm (Ar—H). The MBA molar substitution, ascalculated from the integrals at 7.1-7.5 ppm (Ar—H) and 1.7-2 ppm(HA-acetamide), was 10%.

Example 16 HA-DVS Reaction with 4-methylbenzenethiol (HA-DVS10-MBT)

0.5 g vinyl sulfone derivatized HA (approx. 8%, as per Example 14) wasadded to 27.5 g DI water in a 250 mL round bottom flask. The solutionwas stirred for about 4 hours at room temperature. About 15.98 gdenatured ethanol was added and the mixture was stirred for about 18 hrsat which point the material had dissolved. The flask was then purgedwith nitrogen and then placed in a water bath (temp=30±2° C.). 0.074 g4-methylbenzenethiol (MBT) in about 1.78 g ethanol was then added to thesolution of derivatized HA. The pH of the reaction mixture was adjustedto about 9.5 using 0.25 M NaOH. The solution was stirred for 4 hoursafter which the pH was adjusted to about 7 using 0.25 M HCl. About 0.25g NaCl was added to the reaction solution. The solution was stirreduntil the NaCl had dissolved. 150 mL cold acetone was slowly added tothe solution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. The precipitate waswashed 4 times with 25 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D₂O and the¹H-NMR spectrum was measured. The presence of MBT substitution wasevidenced by peaks at 2.3-2.5 ppm (Ar—CH₃), 7.2-7.6 ppm (Ar—H). The MBTmolar substitution, as calculated from the integrals at 7.1-7.5 ppm(Ar—H) and 1.7-2 ppm (HA-acetamide), was 5.0%.

Example 17 HA-DVS Reaction with 4-methoxy-α-toluenethiol (HA-DVS10-MTT)

0.5 g vinyl sulfone derivatized HA (approx. 8%, as per Example 14) wasadded to 27.5 g DI water in a 250 mL round bottom flask. The solutionwas stirred for about 4 hours at room temperature. 16 g denaturedethanol was added and the mixture was stirred for about 18 hrs at whichpoint the material had dissolved. The flask was then purged withnitrogen and then placed in a water bath (temp=30±2° C.). About 0.092 g4-methoxy-α-toluenethiol (MTT) in 1.78 g ethanol was then added to thesolution of derivatized HA. The pH of the reaction mixture was adjustedto about 9.5 using 0.25 M NaOH. The solution was stirred for 4 hoursafter which the pH was adjusted to about 7 using 0.25 M HCl. 0.25 g NaClwas added to the reaction solution. The solution was stirred until theNaCl had dissolved. 150 mL cold acetone was slowly added to thesolution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. The precipitate waswashed 4 times with 25 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D₂O and the¹H-NMR spectrum was measured. The presence of MTT substitution wasevidenced by peaks at 6.7-7.0 ppm (Ar—H), 7.1-7.3 ppm (Ar—H). The MTTmolar substitution, as calculated from the integrals at 7.1-7.3 ppm(Ar—H) and 1.7-2 ppm (HA-acetamide), was 10.2%.

Example 18 HA-DVS reaction with thiophenol (HA-DVS10-thiophenol)

0.5 g vinyl sulfone derivatized HA (approx. 8%, as per Example 14) wasadded to 27.5 g DI water in a 250 mL round bottom flask. The solutionwas stirred for about 4 hours at room temperature. 16 g denaturedethanol was added and the mixture was stirred for about 18 hrs at whichpoint the material had dissolved. The flask was then purged withnitrogen and then placed in a water bath (temp=30±2° C.). 0.066 gthiophenol in 1.78 g ethanol was then added to the solution ofderivatized HA. The pH of the reaction mixture was adjusted to about 9.5using 0.25 M NaOH. The solution was stirred for 4 hours after which thepH was adjusted to about 7 using 0.25 M HCl. About 0.25 g NaCl was addedto the reaction solution. The solution was stirred until the NaCl haddissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. The precipitate was isolatedusing vacuum filtration. The precipitate was washed 4 times with 25 mLethanol in such a manner that the filter funnel did not run dry. Theprecipitate was dried under vacuum at room temperature. A sample of thematerial was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thepresence of thiophenol substitution was evidenced by peaks at 7.1-7.5ppm (Ar—H). The thiophenol molar substitution, as calculated from theintegrals at 7.1-7.5 ppm (Ar—H) and 1.7-2 ppm (HA-acetamide), was 7.6%.

Examples 19A and 19B Effect of pH on HA-DVS Reaction with 1-pentanethiol(HA-DVS10-pent)

In each of Examples 19A and 19B, 0.5 g vinyl sulfone derivatized HA(approx. 7.5%, as per Example 14) was added to 27.5 g DI water in a 250mL round bottom flask. The solution was stirred for about 4 hours atroom temperature. 16 g denatured ethanol was added and the mixture wasstirred for about 18 hrs at which point the material had dissolved. Theflask was then purged with nitrogen and then placed in a water bath(temp=30±2° C.). About 0.062 g pentanethiol in about 1.78 g ethanol wasthen added to the solution of derivatized HA. In Example 19A, the pH wasadjusted to about 8.5 while in Example 19B, the pH of the reactionmixture was adjusted to about 9.4 using 0.25 M NaOH. For both reactions,the solution was stirred for 4 hours after which the pH was adjusted toabout 7 using 0.25 M HCl. 0.25 g NaCl was added to the reactionsolution. The solution was stirred until the NaCl had dissolved. 150 mLcold acetone was slowly added to the solution. The reaction mixture wasstirred for 1.5 hours. The precipitate was isolated using vacuumfiltration. The precipitate was washed 4 times with 25 mL ethanol insuch a manner that the filter funnel did not run dry. The precipitatewas dried under vacuum at room temperature. A sample of the material wasdissolved in D₂O and the ¹H-NMR spectrum was measured. The presence ofpentanethiol substitution was evidenced by peaks at 0.6-0-8 ppm (CH₃—),1.2-1.6 ppm (—CH₂—), 2.4-2.6 ppm (—CH₂—S—) and 2.7-2.9 ppm (—S—CH2-).The pentanethiol molar substitution, as calculated from the integrals at2.3-2.7 ppm (pent-CH2-S—) and 1.7-2 ppm (HA-acetamide), was 3.9% for pH8.5 reaction and 6.7% for pH 9.5 reaction.

Example 20 DVS Modified HA-(DVS12—800 kDa)

3.5 g sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) was added to aglass 4 L reaction kettle. The lid, overhead stirrer and anchor impellorwere attached to the reaction kettle. 350 g deionized water was added tothe kettle. The solution was stirred at about 750 rpm for about 18 hrs.About 233 g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be 12.91. A freshly prepared solution of 15.5 g divinyl sulfonein 92 g of DI water was then rapidly added to the stirring solution.After 3.25 minutes, 63 g of a 1M HCl solution was added to the reactionmixture. Either 1M NaOH or 1M HCl was then added dropwise as neededuntil the solution pH was between 5 and 7. About 8.4 g NaCl was thenadded to the solution. Once the NaCl had dissolved, 1.5 L acetone wasslowly added over a period of 30 minutes. The suspension was stirred forabout 3 hours. 300 mL ethanol (Ethanol, Alcohol Reagent, Denaturedanhydrous 94-96%) was added and the solution was stirred for about 30minutes. The precipitate was filtered under vacuum using a sinteredglass funnel through a 0.22 μm PTFE filter membrane. Once all thesolution had been filtered, the vacuum was disconnected and 150 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temp in a vacuum oven. Thepercent substitution, as determined according to the procedure describedin Example 1, was found to be about 22.2%.

Example 21 HA-DVS Reaction with 3-Mercaptopropionic acid (HA-DVS12-MPA)

1.0 g vinyl sulfone derivatized HA (approx. 22%, as per Example 20) wasadded to 100 g DI water in a 250 mL round bottom flask. The solution wasstirred overnight until the material had dissolved. The flask was thenpurged with nitrogen. 0.106 g 3-mercaptopropionic acid (MPA) was addedto the solution. After the MPA had dissolved, the pH was adjusted toabout 9 using 0.25 M NaOH. The solution was stirred for 4 hours afterwhich the pH was adjusted to about 7 using 0.25 M HCl. 2.4 g NaCl wasadded to the reaction solution. The solution was stirred until the NaClhad dissolved. 300 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. 50 mL ethanol was added andthe resultant mixture was stirred for 15 minutes. The precipitate wasisolated using vacuum filtration. The precipitate was washed 4 timeswith 50 mL ethanol in such a manner that the filter funnel did not rundry. The precipitate was dried under vacuum at room temperature. Asample of the material was dissolved in D₂O and the ¹H-NMR spectrum wasmeasured. The presence of MPA substitution was evidenced by peaks at2.4-2.6 ppm (—CH₂—COOH), 2.7-2.8 ppm (—CH₂—S—) and 2.9-3.1 ppm(—S—CH₂—). The MPA substitution, as calculated from the integrals at2.4-2.6 ppm (MPA-CH₂) and 1.7-2 ppm (HA-acetamide), was 20.6%.

Example 22 DVS modified HA-(DVS14—800 kDa)

3.5 g sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) was added to aglass 4 L reaction kettle. The lid, overhead stirrer and anchor impellorwere attached to the reaction kettle. 350 g deionized water was added tothe kettle. The solution was stirred at about 750 rpm for about 18 hrs.233 g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be 12.87. A freshly prepared solution of 15.5 g divinyl sulfonein 92 g of DI water was then rapidly added to the stirring solution.After 6 minutes, 63 g of a 1M HCl solution was added to the reactionmixture. Either 1M NaOH or 1M HCl was then added dropwise as neededuntil the solution pH was between 5 and 7. About 8.4 g NaCl was thenadded to the solution. Once the NaCl had dissolved, 1.5 L acetone wasslowly added over a period of 30 minutes. The suspension was stirred forabout 3 hours. 3 00 mL ethanol (Ethanol, Alcohol Reagent, denaturedanhydrous 94-96%) was added and the solution was stirred for about 30minutes. The precipitate was filtered under vacuum using a sinteredglass funnel through a 0.22 μm PTFE filter membrane. 150 mL ethanol wasused to rinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times. The productwas dried under vacuum at room temp in a vacuum oven. The percentsubstitution, as determined by the procedure described in Example 1, wasfound to be 31.4%.

Example 23 HA-DVS Reaction with 2-mercaptobenzoic acid (HA-DVS14-MBA)

1.0 g vinyl sulfone derivatized HA (approx. 31%, as per Example 22) wasadded to 55 g DI water in a 500 mL round bottom flask. The solution wasstirred for about 1 hour at room temperature. 32 g denatured ethanol wasadded and the mixture was stirred for about 18 hrs at which point thematerial had dissolved. The flask was then purged with nitrogen and thenplaced in a water bath (temp=30±2° C.). 0.554 g 2-mercaptobenzoic acid(MBA) in 3.55 g ethanol was then added to the derivatized HA solution.The pH of the reaction mixture was adjusted to about 9 using 0.25 MNaOH. The solution was stirred for 4 hours after which the pH wasadjusted to about 7 using 0.25 M HCl. 1.32 g NaCl was added to thereaction solution. The solution was stirred until the NaCl haddissolved. 300 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. The precipitate was isolatedusing vacuum filtration. The precipitate was washed 3 times with 50 mLethanol in such a manner that the filter funnel did not run dry. Theprecipitate was dried under vacuum at room temperature. A sample of thematerial was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thepresence of MBA substitution was evidenced by peaks at 7.1-7.5 ppm(Ar—H) [FIG. 4]. The MBA molar substitution, as calculated from theintegrals at 7.1-7.5 ppm (Ar—H) and 1.7-2 ppm (HA-acetamide) was 35%.

Example 24 HA-DVS Reaction with mercaptosuccinic acid (HA-DVS14-MSA)

1.0 g vinyl sulfone derivatized HA (approx. 31%, as per Example 22) wasadded to 100 g DI water in a 500 mL round bottom flask. The solution wasstirred for about 18 hrs at which point the material had dissolved. Theflask was then purged with nitrogen. 0.18 g mercaptosuccinic acid (MSA)was then added to the derivatized HA solution. The pH of the reactionmixture was adjusted to about 9 using 0.25 M NaOH. The solution wasstirred for 4 hours after which the pH was adjusted to about 7 using0.25 M HCl. About 2.4 g NaCl was added to the reaction solution. Thesolution was stirred until the NaCl had dissolved. 300 mL cold acetonewas slowly added to the solution. The reaction mixture was stirred for1.5 hours. 50 mL ethanol was added and the mixture was stirred for 15minutes. The precipitate was isolated using vacuum filtration. Theprecipitate was washed 4 times with 50 mL ethanol in such a manner thatthe filter funnel did not run dry. The precipitate was dried undervacuum at room temperature. A sample of the material was dissolved inD₂O and the ¹H-NMR spectrum was measured. The presence of MSAsubstitution was evidenced by peaks at 2.3 to 3.1 ppm. The MSA molarsubstitution, as calculated from the integrals at 3.0 ppm and 1.7-2 ppm(HA-acetamide) was 33%.

Example 25 HA-DVS reaction with 9-mercapto-1-nonanol (HA-DVS14-nonanol)

1.0 g vinyl sulfone derivatized HA (approx. 31%, as per Example 22) wasadded to 55 g DI water in a 500 mL round bottom flask. The solution wasstirred for about 1 hour at room temperature. 32 g denatured ethanol wasadded and the mixture was stirred for about 18 hrs at which point thematerial had dissolved. The flask was then purged with nitrogen and thenplaced in a water bath (temp=30±2° C.). 0.63 g 9-mercapto-1-nonanol in3.55 g ethanol was then added to the solution of derivatized HA. The pHof the reaction mixture was adjusted to about 9 using 0.25 M NaOH. Thesolution was stirred for 4 hours after which the pH was adjusted toabout 7 using 0.25 M HCl. 1.32 g NaCl was added to the reactionsolution. The solution was stirred until the NaCl had dissolved. 300 mLcold acetone was slowly added to the solution. The reaction mixture wasstirred for 1.5 hours. The precipitate was isolated using vacuumfiltration. The precipitate was washed 3 times with 50 mL ethanol insuch a manner that the filter funnel did not run dry. The precipitatewas dried under vacuum at room temperature. A sample of the material wasdissolved in D₂O and the ¹H-NMR spectrum was measured. The presence ofnonanol substitution was evidenced by peaks at 1.1-1.8 ppm (—CH₂—),2.5-2.8 ppm (—CH₂—S—) and 2.9-3.1 ppm (—S—CH₂—. The nonanol molarsubstitution, as calculated from the integrals at 2.5-2.8 ppm (—CH₂—S—)and 1.7-2 ppm (HA-acetamide), was 37.5%.

Example 26 DVS Reaction with 3-Mercaptopropionic Acid Derivatized HA[HA-DVS12-MPA] (HA-MPA-DVS)

0.75 g MPA derivatized sodium hyaluronate (see Example 21) was added toa glass 4 L reaction kettle. The lid, overhead stirrer and anchorimpellor were attached to the reaction kettle. 75 g deionized water wasadded to the kettle. The solution was stirred at about 750 rpm for about18 hrs. 50 g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured to be 12.85. A freshlyprepared solution of 3.3 g divinyl sulfone in 20 g of DI water was thenrapidly added to the stirring solution. After 3.25 minutes, 13.5 g of a1M HCl solution was added to the reaction mixture. Either 1M NaOH or 1MHCl was then added dropwise as needed until the solution pH was between5 and 7. About 1.8 g NaCl was then added to the solution. Once the NaClhad dissolved, 300 mL acetone was slowly added over a period of 30minutes. The suspension was stirred for about 3 hours. 50 mL ethanol wasadded and the solution was stirred for about 30 minutes. The precipitatewas filtered under vacuum using a sintered glass funnel. Once all thesolution had been filtered, the vacuum was disconnected and 150 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temperature in a vacuum oven.The percent vinyl sulfone substitution, as determined according to theprocedure described in Example 1, was found to be 10.1%.

Example 27 HA-MPA-DVS Reaction with 3-Mercaptopropionic acid(HA-MPA{circumflex over ( )}2-DVS{circumflex over ( )}2)

0.5 g MPA/vinyl sulfone derivatized HA (see Example 26) was added to 50g DI water in a 250 mL round bottom flask. The solution was stirredovernight until the material had dissolved. The flask was then purgedwith nitrogen. 0.048 g 3-mercaptopropionic acid (MPA) was added to thesolution. After the MPA had dissolved, the pH was adjusted to about 9using 0.25 M NaOH. The solution was stirred for 4 hours after which thepH was adjusted to about 7 using 0.25 M HCl. 1.2 g NaCl was added to thereaction solution. The solution was stirred until the NaCl haddissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. 25 mL ethanol was added andthe resultant mixture was stirred for 15 minutes. The precipitate wasisolated using vacuum filtration. The precipitate was washed 4 timeswith 25 mL ethanol in such a manner that the filter funnel did not rundry. The precipitate was dried under vacuum at room temperature. Asample of the material was dissolved in D₂O and the ¹H-NMR spectrum wasmeasured. The presence of MPA substitution was evidenced by peaks at2.4-2.6 ppm (—CH₂—COOH), 2.7-2.8 ppm (—CH₂—S—) and 2.9-3.1 ppm(—S—CH₂—). The MPA substitution, as calculated from the integrals at2.4-2.6 ppm (MPA-CH₂) and 1.7-2 ppm (HA-acetamide) was 33%.

Example 28 DVS Reaction with thiophenol Derivatized HA[HA-10-thiophenol-DVSA2]

0.323 g MPA derivatized sodium hyaluronate (see Example 18) was added toa 250 mL round bottom flask. The overhead stirrer and anchor impellorwere put in place. 4.62 g deionized water was added to the kettle. Thesolution was stirred at about 750 rpm for about 18 hrs. 23.48 g of a0.25 M NaOH solution was added to the dissolved sodium hyaluronatederivative. The pH of the solution was measured to be 12.82. A freshlyprepared solution of 0.775 g divinyl sulfone in 4.62 g of DI water wasthen rapidly added to the stirring solution. After 2.5 minutes, 6.15 gof a 1M HCl solution was added to the reaction mixture. Either 0.25MNaOH or 1M HCl was then added dropwise as needed until the solution pHwas between 5 and 7. About 0.858 g NaCl was then added to the solution.Once the NaCl had dissolved, 125 mL acetone was slowly added over aperiod of 5 minutes. The suspension was stirred for about 3 hours. 25 mLethanol was added and the solution was stirred for about 30 minutes. Theprecipitate was filtered under vacuum using a sintered glass funnel.Once all the solution had been filtered, 100 mL ethanol was used torinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times. The productwas dried under vacuum at room temperature in a vacuum oven. The percentvinyl sulfone substitution, as determined according to the proceduredescribed in Example 1, was found to be 13.6%. The thiophenol peaks wereobserved in the 7.2-7.5 ppm.

Example 29 Crosslinking with a PEG-dithiol Compound

Approx. 108 mg HA-10-thiophenol-DVSA2 [see Example 28] was weighed intoa 20 mL glass scintillation vial. 7.2 mL deionized water was added andthe sample was allowed to dissolve overnight. Approx. 65.64 mgPEG[3400]-(SH)2 [Sigma Aldrich] was added to a glass scintillation vial.2.6 mL deionized water was added to the vial and the sample was mixeduntil dissolved. The PEG[3400]-(SH)2 solution was added to theHA-10-thiophenol-DVSA2 solution and the pH of the resultant solution wasadjusted to greater than pH 10 (pH=10.71) using 0.25 M NaOH. Thesolution turned to a gel state.

Example 30 Crosslinking with Trimethylolpropanetris(3-mercaptopropionate) [TMP-SH]

Approx. 109 mg HA-10-thiophenol-DVSA2 [see Example 28] was weighed intoa 20 mL glass scintillation vial. 7.29 mL deionized water was added andthe sample was allowed to dissolve overnight. Approx. 6.1 mgtrimethylolpropane tris(3-mercaptopropionate) [TMP-SH] [Sigma Aldrich]was added to a glass scintillation vial. 213 mL deionized water wasadded to the vial and the sample. The TMP-SH mixture was added to theHA-10-thiophenol-DVSA2 solution and the pH of the resultant solution wasadjusted to greater than pH 11 (pH=11.31) using 0.25 M NaOH. Thesolution turned to a gel state.

Example 31 DVS Reaction with HA [HA-DVS-16-2]

10 g sodium hyaluronate (1.4 m3/kg, approx. 800 kD) was added to a glass4 L reaction kettle. The lid, overhead stirrer and anchor impellor wereattached to the reaction kettle. 1000 g deionized water was added to thekettle. The solution was stirred at about 300 rpm for about 18 hrs.166.5 g of a 1 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be greater than 12.5. A freshly prepared solution of 44.2 gdivinyl sulfone in 250 g of DI water was then rapidly added to thestirring solution. After 8 minutes, 170 g of a 1M HCl solution was addedto the reaction mixture. Either 1M NaOH or 1M HCl was then addeddropwise as needed until the solution pH was between 5 and 7. About 24 gNaCl was then added to the solution. Once the NaCl had dissolved, 3 Lacetone was slowly added over a period of 40 minutes. The suspension wasstirred for about 3 hours. 500 mL ethanol was added and the solution wasstirred for about 30 minutes. The precipitate was filtered under vacuumusing a sintered glass funnel through a 0.22 μm PTFE filter membrane.Once all the solution had been filtered, the vacuum was disconnected and200 mL ethanol was used to rinse the precipitate. The ethanol was thenremoved by vacuum filtration. This process was repeated an additional 3times. The product was dried under vacuum at room temp in a vacuum oven.The percent substitution, as determined by the procedure described inExample 1, was found to be 72%.

Example 32 HA-DVS Reaction with 3-mercapto-1-propanesulfonate[HA-DVS-16-2-SMPS]

2.0 g vinyl sulfone derivatized HA (as per Example 31) was added to 200g DI water in a 1 L reaction vessel. The solution was stirred (approx.300 rpm) overnight until the material had dissolved. The flask was thenpurged with nitrogen. 1.28 g sodium-3-mercapto-1-propanesulfonate (SMPS) was added to the solution. After the SMPS had dissolved, the pH wasadjusted to about 9 using 0.25 M NaOH. The solution was stirred for 4hours after which the pH was adjusted to about 7 using 0.25 M HCl. 4.8 gNaCl was added to the reaction solution. The solution was stirred untilthe NaCl had dissolved. 300 mL cold acetone was slowly added to thesolution. The reaction mixture was stirred for 1.5 hours. 75 mL ethanolwas added and the resultant mixture was stirred for 15 minutes. Theprecipitate was isolated using vacuum filtration. The precipitate waswashed 4 times with 75 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D₂O and the¹H-NMR spectrum was measured. The presence of SMPS substitution wasevidenced by peaks at 2.0-2.1 ppm (—CH₂—), 2.5-2.7 ppm (—CH₂—S—) and2.8-3.0 ppm (—S—CH₂—). The SPMS substitution, as calculated from theintegrals at 2.5-2.7 ppm (SMPS-CH₂—S—) and 1.7-2 ppm (HA-acetamide), was82%.

Example 33 Crosslinking with divinyl sulfone—Single Component

Approx. 105 mg HA-DVS-16-2-SMPS [see Example 32] was weighed into a 20mL glass scintillation vial. Approx. 2.3 mL deionized water was added tothe vial and the sample was allowed to dissolve overnight. An additional0.26 g DI water was added and the sample was mixed. The pH of thesolution was adjusted to approximately pH 13 using 1M NaOH.Approximately 26 μl divinyl sulfone was added to the reaction mixture.The sample was mixed by vortexing and the reaction mixture was left atroom temperature until a gel had formed. The gel was removed from thevial and placed in approx. 500 mL deionized water for 1 hour. The waterwas changed 2 times following an incubation time between 40 to 70minutes. The water excess water was removed, the gel was transferred toa plastic container and the gel was frozen at −80° C. and then waslyophilized to produce a porous foam structure.

Example 34 Crosslinking with divinyl sulfone—Two Component

100 mg HA-DVS-16-2-SMPS [see Example 32] and 100 mg HA-DVS14-nonanol(see Example 25]) is weighed into a 20 mL glass scintillation vial. 5 mLdeionized water is added to the vial and the sample is allowed todissolve overnight. The pH of the solution is adjusted to approximatelypH 13 using 1M NaOH. 52 μl divinyl sulfone is added to the reactionmixture. The sample is mixed by vortexing and the reaction mixture isleft at room temperature until a gel forms. The gel is removed from thevial and is placed in 500 mL deionized water for 1 hour. The water ischanged 2 times following incubation times between about 40 to 70minutes. The water excess water is removed, the gel is transferred to aplastic container. A portion of the gel is frozen at −80° C. and is thenlyophilized to produce a porous foam structure.

Example 35 Crosslinked Particles

Particles of the crosslinked hydrogels (representative examples includeExamples 29, 30, 33 and 34) are prepared by passing the crosslinkedderivatized polyhydric polymer gel composition through a mesh. Thecrosslinked gel is transferred to a 20 mL syringe. 5 mL saline or 10mg/mL hyaluronic acid (approx. 800 kDa) is added to the syringe. Theplunger is inserted into the syringe and the gel is extruded through amesh (mesh with a pore size of approximately 500 μm that is held in aPolycarbonate Filter Holder, 25 mm). The extrusion process is repeatedto produce gel particles.

Example 36 DVS Modified HA-(DVS18—800 kDa)

3.5 g sodium hyaluronate (IV=1.4 m³/kg, MW approx. 800 kD) was added toa glass 4 L reaction kettle. The lid, overhead stirrer and anchorimpellor were attached to the reaction kettle. 350 g deionized water wasadded to the kettle. The solution was stirred at about 750 rpm for about18 hrs. 233 g of a 0.25 M NaOH solution was added to the dissolvedsodium hyaluronate. The pH of the solution was measured after 2 min andwas found to be 12.92. A freshly prepared solution of 15.5 g divinylsulfone in 92 g of DI water was then rapidly added to the stirringsolution. After 15 minutes, 63 g of a 1M HCl solution was added to thereaction mixture. Either 1M NaOH or 1M HCl was then added dropwise asneeded until the solution pH was between 5 and 7. About 8.4 g NaCl wasthen added to the solution. Once the NaCl had dissolved, 1.5 L acetonewas slowly added over a period of 30 minutes. The suspension was stirredfor about 3 hours. 300 mL ethanol (Ethanol, Alcohol Reagent, Denaturedanhydrous 94-96%) was added and the solution was stirred for about 30minutes. The precipitate was filtered under vacuum using a sinteredglass funnel through a 0.22 μm PTFE filter membrane. Once all thesolution had been filtered, the vacuum was disconnected and 150 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temperature in a vacuum oven.The percent substitution, as determined by the procedure described inExample 1, was found to be 71.3%.

Example 37 HA-DVS Reaction with 3-Mercaptopropionic Acid (HA-DVS18-MPA)

1.0 g vinyl sulfone derivatized HA (approx. 71%, as per Example 36) wasadded to 100 g DI water in a 500 mL round bottom flask. The solution wasstirred at 300 rpm overnight so that all of the material had dissolved.The flask was then purged with nitrogen. 0.25 g 3-mercaptopropionic acid(MPA) was added to the solution. After the MPA had dissolved, the pH wasadjusted to about 9 using 0.25 M NaOH. The solution was stirred for 4hours after which the pH was adjusted to about 7 using 0.25 M HCl. 2.4 gNaCl was added to the reaction solution. The solution was stirred untilthe NaCl had dissolved. 300 mL cold acetone was slowly added to thesolution. The reaction mixture was stirred for 1.5 hours. 50 mL ethanolwas added and the resultant mixture was stirred for 15 minutes. Theprecipitate was isolated using vacuum filtration. The precipitate waswashed 4 times with 50 mL ethanol in such a manner that the filterfunnel did not run dry. The precipitate was dried under vacuum at roomtemperature. A sample of the material was dissolved in D2O and the1H-NMR spectrum was measured. The presence of MPA substitution wasevidenced by peaks at 2.4-2.6 ppm (—CH₂—COOH), 2.7-2.8 ppm (—CH₂—S—) and2.9-3.1 ppm (—S—CH₂—). The MPA substitution, as calculated from theintegrals at 2.4-2.6 ppm (MPA-CH₂) and 1.7-2 ppm (HA-acetamide), was79.4%.

Example 38 DVS Reaction with 3-Mercaptopropionic Acid Derivatized HA[HA-18-MPA-DVS]

0.75 g MPA derivatized sodium hyaluronate (see Example 37) was added toa glass 4 L reaction kettle. The lid, overhead stirrer and anchorimpellor were attached to the reaction kettle. 75 g deionized water wasadded to the kettle. The solution was stirred at about 200 rpm for about18 hrs. 50 g of a 0.25 M NaOH solution was added to the dissolved sodiumhyaluronate. The pH of the solution was measured to be 12.70. A freshlyprepared solution of 3.3 g divinyl sulfone in 20 g of DI water was thenrapidly added to the stirring solution. After 15 minutes, 13.5 g of a 1MHCl solution was added to the reaction mixture. Either 1M NaOH or 1M HClwas then added dropwise as needed until the solution pH was between 5and 7. About 1.8 g NaCl was then added to the solution. Once the NaClhad dissolved, 300 mL acetone was slowly added over a period of 30minutes. The suspension was stirred for about 3 hours. 50 mL ethanol wasadded and the solution was stirred for about 30 minutes. The precipitatewas filtered under vacuum using a sintered glass funnel. Once all thesolution had been filtered, the vacuum was disconnected and 150 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temperature in a vacuum oven.The percent vinyl sulfone substitution, as determined according to theprocedure described in Example 1, was found to be 66%.

Example 39 HA-MPA-DVS Reaction with 3-Mercaptopropionic Acid(HA-MPAA2-DVSA2)

0.5 g MPA/vinyl sulfone derivatized HA (see Example 38) was added to 50g DI water in a 250 mL round bottom flask. The solution was stirredovernight, during which time the material dissolved. The flask was thenpurged with nitrogen. 0.175 g 3-mercaptopropionic acid (MPA) was addedto the solution. After the MPA had dissolved, the pH was adjusted toabout 9 using 0.25 M NaOH. The solution was stirred for 4 hours afterwhich the pH was adjusted to about 7 using 0.25 M HCl. 1.2 g NaCl wasadded to the reaction solution. The solution was stirred until the NaClhad dissolved. 150 mL cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. 25 mL ethanol was added andthe resultant mixture was stirred for 15 minutes. The precipitate wasisolated using vacuum filtration. The precipitate was washed 4 timeswith 25 mL ethanol in such a manner that the filter funnel did not rundry. The precipitate was dried under vacuum at room temperature. Asample of the material was dissolved in D₂O and the ¹H-NMR spectrum wasmeasured. The presence of MPA substitution was evidenced by peaks at2.4-2.6 ppm (—CH₂—COOH), 2.7-2.8 ppm (—CH₂—S—) and 2.9-3.1 ppm(—S—CH₂—). The MPA substitution, as calculated from the integrals at2.4-2.6 ppm (MPA-CH2) and 1.7-2 ppm (HA-acetamide) was 133%.

Example 40 HA-Based Formulations

Various formulations, as shown in Table 1, are prepared where thenumbers listed in Table 1 are weight percent values based on the totalweight of a formulation. Each of the following HA derivatives may beused as the “HA derivative” listed in Table 1: (a) thiophenol derivative(representative example 28), (b) 2-mercaptobenzoic acid derivative(representative example 23), (c) mercaptosuccinic acid derivative(representative example 24), (d) sodium 3-mercapto-1-propanesulfonatederivative (representative example 32) and (e) decanethiol derivative(representative example 11). A portion of the gel samples is cast onto aglass sheet or a piece of release liner and allowed to dry at roomtemperature followed by vacuum drying for at least 12 hours. Thisproduces a film of the formulation. A second portion of the gel samplesare placed in a scintillation vial frozen at −80° C. and are thenlyophilized. The formulations listed in Table 1 can also comprise 1%(w/w) collagen or gelatin as well as other excipients and buffers. Wateror water for injection can be used instead of the saline.

TABLE 1 HA Gellan Pluronics Xantham Formulation derivative gum F127 HPMCCMC PolyAA Chitosan gum PEG400 saline 1 0.3 2 — — — — — — — 97.7 2 0.3 —25 — — — — — — 74.7 3 0.3 — — 0.2 — — — — — 99.5 4 0.3 — — — 0.2 — — — —99.5 5 0.3 — — — — 2.5 — — — 97.2 6 0.3 — — — — — 3 — — 96.7 7 0.3 — — —— — — 2 — 97.7 8 0.3 2 — — — — — — 1 96.7 9 0.3 — 25 — — — — — 1 73.7 100.3 — — 0.2 — — — — 1 98.5 11 0.3 — — — 0.2 — — — 1 98.5 12 0.3 — — — —2.5 — — 1 96.2 13 0.3 — — — — — 3 — 1 95.7 14 0.3 — — — — — — 2 1 96.715 0.2 2 20 — — — — — — 77.8 16 0.2 — 25 0.2 — — — — — 74.6 17 0.2 — 25— — 2   — — — 72.8 18 0.2 — 25 — 2   — — — — 72.8 19 0.2 — 25 — — — 3 —— 71.8 20 0.2 — 25 — — — — 2 — 72.8 21 0.2 2 20 — — — — — 1 76.8 22 0.2— 25 0.2 — — — — 1 73.6 23 0.2 — 25 — — 2   — — 1 71.8 24 0.2 — 25 — 2  — — — 1 71.8 25 0.2 — 25 — — — 3 — 1 70.8 26 0.2 — 25 — — — — 2 1 71.827 0.2 — — — — — — — — 99.8 HPMC = hydroxypropyl methylcellulose CMC =sodium carboxymethylcellulose PolyAA = polyacrylic acid

Example 41 HA-Derivative Formulations

Various formulations, as shown in Table 2, of different HA derivatives,are prepared where the values listed under each ingredient are weightpercent values based on the total weight of the formulation. A portionof the samples are cast onto a glass sheet or a piece of release linerusing a Gardner knife and allowed to dry at room temperature followed byvacuum drying for at least 12 hours. This produces a film of theformulation. A second portion of the gel samples are placed in ascintillation vial frozen at −80° C. and are then lyophilized to producea porous solid matrix. A third portion is kept in the solution/gel form.The formulations listed in Table 2 can also comprise 1% (w/w) collagenor gelatin as well as other excipients and buffers. Water or water forinjection can be used instead of the saline.

TABLE 2 Formulation HA thiophenol 1 MBA MSA SMPS Dec saline 1 — 0.2 — —— — 99.8 2 — — 0.2 — — — 99.8 3 — — — 0.2 — — 99.8 4 — — — 0.2 — 99.8 5— — — 0.2 99.8 6 — 0.1 — 0.1 — 99.8 7 — 0.1 — 0.1 — 99.8 8 — — 0.1 0.1 —99.8 9 — — 0.1 0.1 — 99.8 10 — — 0.1 0.1 99.8 11 — — 0.1 0.1 99.8 12 0.10.1 — — — — 99.8 13 0.1 — 0.1 — — — 99.8 14 0.1 — — 0.1 — — 99.8 15 0.1— — — 0.1 — 99.8 16 0.1 — — — — 0.1 99.8 17 — 0.1 — 0.1 — — 99.8 18 —0.1 — — 0.1 — 99.8 19 — — 0.1 0.1 — — 99.8 20 — — 0.1 — 0.1 — 99.8 21 —— — 0.1 — 0.1 99.8 22 — — — — 0.1 0.1 99.8

Example 42 Biologically Active Agent Incorporation

Biologically active agents that are listed in Table 3 are incorporateddirectly into each of the formulations prepared in Examples 40 and 41.The formulations are prepared in either the gel form, a film form or alyophilized form. Each formulation contains an active agent as listed inTable 3, in the amount as stated on a weight/weight (w/w) basis orunits/gram (U/g) basis or μg/mL basis.

TABLE 3 Amount in final Biologically active agent formulation MitomycinC 0.2% (w/w) Paclitaxel 0.1% (w/w) Chlorhexidine gluconate 2% (w/w)Silver sulfadiazine 1% (w/w) Silver particles (200-400 nm) 1.5% (w/w)Lidocaine 2% (w/w) Bupivacaine 1% (w/w) Triamcinolone acetonide 2% (w/w)Triamcinolone hexacetonide 2% (w/w) Dexamethasone 2% (w/w) Botox 5 U/gBMP-7 0.5 μg/mL Ciproflaxin 6% (w/w)

Example 43 Antibacterial Agent Incorporation

Clindamycin phosphate (1% [w/w]) or metronidazole (1.3% [w/w]) areincorporated directly into the formulations prepared in Example 40 and41. In another set of formulations, a combination of clindamycinphosphate (0.5% [w/w]) or metronidazole (0.5% [w/w]) is incorporateddirectly into the formulations prepared in Example 40 and 41. In anotherset of formulations as prepared in Example 40 and 41, that containeither clindamycin phosphate (1% [w/w]) or metronidazole (1.3% [w/w]),the saline is replaced with a citrate/saline buffer to ensure the pH ismaintained in the 4.5 to 6 pH range. The formulations are prepared ineither the gel form, a film form or a lyophilized form.

Example 44 Rehydration with Biologically Active Agent

Lyophilized products can be prepared from formulations described inExamples 29, 30, 33, 34, 40 and 41. The lyophilized derivatizedpolyhydric polymer composition is rehydrated with either BMP-7 (5 μg/mL)or Botox (5 U/mL). The resultant gel is used as a gel and is applied tothe tissue of a subject through topical application or throughinjection.

Example 45 Incorporation of Cells into Gel

Approx. 100 mg of a derivatized hyaluronic acid that contains residualvinyl sulfone groups (representative example as described in Example 28)is weighed into a 20 mL glass scintillation vial. 7 mL saline is addedand the sample is allowed to dissolve overnight. Approx. 64 mgPEG[3400]-(SH)2 (Sigma Aldrich, St. Louis, Mo., US) is added to a glassscintillation vial. 1.3 mL saline is added to the vial and the sample ismixed until dissolved. The PEG[3400]-(SH)2 solution is added to thederivatized HA sample solution and the pH of the resultant solution isadjusted to pH 8.5 a using 0.25 M NaOH. An aliquot of freshlytrypsinized hMSCs cell suspension is mixed with the HA/PEG solution toprovide a final cell concentration of 1, 5, 10 and 20×10⁶ cells/mL. Thesamples are aliquoted into a 12 well plate. The samples are allowed togel for 20 min in a 37° C. incubator. 2 mL fresh media is then added toeach well.

Example 46 Incorporation of Gel into a Scaffold

Approx. 100 mg of a derivatized hyaluronic acid that contains residualvinyl sulfone groups (representative example as described in Example 28)is weighed into a 20 mL glass scintillation vial. 7 mL saline is addedand the sample is allowed to dissolve overnight. Approx. 64 mgPEG[3400]-(SH)2 [Sigma Aldrich] is added to a glass scintillation vial.1.3 mL saline is added to the vial and the sample is mixed untildissolved. The PEG[3400]-(SH)2 solution is added to the derivatized HAsample solution and the pH of the resultant solution is adjusted to pH8.5 a using 0.25 M NaOH. The resultant solution is aliquoted onto aporous scaffold and is allowed to soak into the scaffold. Once thescaffold is saturated, the scaffold is placed in an incubator (37° C.)until the crosslinking reaction is completed. The porous scaffolds usedfor gel incorporation are an electrospun polydioxanone fabric and a3D-printed polylactide scaffold with pores of approximately 200-500 μm.

Example 47 Incorporation of Gel/Cell Matrix into a Scaffold

A gel/cell matrix, as prepared in Example 45, is aliquoted onto a porousscaffold prior to gelation and is allowed to soak into the scaffold.Once the scaffold is saturated, the scaffold is placed in an incubator(37° C.) until the crosslinking reaction is completed. The porousscaffolds used for gel incorporation are an electrospun polydioxanonefabric and a 3D-printed polylactide-co-glycolide scaffold with pores ofapproximately 200-500 μm. The porous scaffold/hydrogel/cell complexesare placed in a well of a 12 well culture plate that contains freshmedia

Example 48 Electrospinning of HA-Derivative

A 15 mg/mL solution of a hyaluronic acid derivative is prepared byadding approximately 90 mg hyaluronic acid derivative to 3 mL deionizedwater. The sample is allowed to dissolve overnight. 3 mLdimethylformamide (DMF) is added to the sample and the sample isvortexed several times over a 30 minute period. The solution istransferred to a 5 mL syringe that has a needle tip with a 0.3 mminternal diameter. The syringe pump is set at 60 mL/min. The appliedvoltage is set at 22 kV with the distance from the tip to the collectorbeing 15 cm. A piece of heavy aluminum foil is connected to the groundwire and the foils is submerged into a shallow bath that containsethanol. The resulting electrospun derivatized polyhydric polymercomposition is carefully removed and the sample is placed in the vacuumoven to remove residual solvent.

Example 49 Formation of Shaped Hydrogels

Approx. 100 mg of a derivatized hyaluronic acid that contained residualvinyl sulfone groups (representative example as described in Example 28)is weighed into a 20 mL glass scintillation vial. 7 mL saline is addedand the sample is allowed to dissolve overnight. Approx. 64 mgPEG[3400]-(SH)2 [Sigma Aldrich] is added to a glass scintillation vial.1.3 mL saline is added to the vial and the sample is mixed untildissolved. The PEG[3400]-(SH)₂ solution is added to the derivatized HAsample solution and the pH of the resultant solution is adjusted to pH 8a using 0.25 M NaOH. 30 mg of a drug (e.g., dexamethasone, triamcinoloneacetonide, budesonide, flunisolide, ciproflaxin) was mixed into thesolution. Using a syringe with a piece of silastic tubing of a knowndiameter attached to the needle, the solution is drawn up into thesilastic tubing. Once the tubing is almost filled, the open end is bentover and held closed with a clamp. The tube is placed in an incubator(37° C.) overnight to complete the crosslinking reaction. The clamp isthen removed and the gel in the tubing is dried in a vacuum oven. Thedried crosslinked derivatized polyhydric polymer composition is removedfrom the tubing by carefully slicing the tubing. The dried crosslinkedderivatized polyhydric polymer composition is then cut to the desiredlength (e.g. 2 to 4 mm and 1 to 2 cm). The solution preparation can use60% (v/v) water and 40% (ethanol) in place of 100% water. Driedcrosslinked derivatized polyhydric polymer composition without the drugare also prepared.

Example 50 Synthesis of divinyl sulfone Derivatized HA—DifferentMolecular Weights

The synthesis of divinyl sulfone derivatized HA using different startingHA molecular weights were performed using a similar method as describedin Example 1. The specific molecular weights, reaction conditions andvinyl sulfone substitution obtained are as set forth in Table 4:

TABLE 4 Rxn1 Rxn1 Rxn3 Approx. Mw (kD) 2,300 200 510 HA (g) 2.5 5 5Water (g) 500 500 500 DVS (g) 10.6 21.2 21.2 DVS water (g) 66 132 132Stir speed (rpm) 200-250 200-250 200-250 Reaction pH >12.5 >12.5 >12.5Reaction time (min) 1.25 1.25 1.25 NaCl (g) 4 12 12 Acetone (mL) 20002000 1750 Ethanol (mL) 200 400 400 Ethanol wash (mL) 100 200 200Substitution (%) 8.1 6.4 9.3

Example 51 DVS reaction with HA-MSA (HA-DVS12-MSA-DVS)

0.75 g MPA derivatized sodium hyaluronate (see Example 24) was added toa 250 round bottom flask. An overhead stirrer and anchor impellor wereattached to the reaction flask. 75 g deionized water was added to thekettle. The solution was stirred at about 750 rpm for about 18 hrs. 50 gof a 0.25 M NaOH solution was added to the dissolved sodium hyaluronate.The pH of the solution was measured to be about 12.8. A freshly preparedsolution of 3.3 g divinyl sulfone in 20 g of DI water was then rapidlyadded to the stirring solution. After 3.25 minutes, 13.5 g of a 1M HClsolution was added to the reaction mixture. Either 1M NaOH or 1M HCl wasthen added dropwise as needed until the solution pH was between 5 and 7.About 1.8 g NaCl was then added to the solution. Once the NaCl haddissolved, 300 mL acetone was slowly added over a period of 30 minutes.The suspension was stirred for about 3 hours. 50 mL ethanol was addedand the solution was stirred for about 30 minutes. The precipitate wasfiltered under vacuum using a sintered glass funnel. Once all thesolution had been filtered, the vacuum was disconnected and 50 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 times.The product was dried under vacuum at room temperature in a vacuum oven.The percent vinyl sulfone substitution, as determined according to theprocedure described in Example 1, was found to be 47.8%.

Example 52 MPA reaction with HA-MSA-DVS (HA-DVS12-MSA-MPA)

0.5 g HA-MSA-DVS (Example 51) was added to 50 g DI water in a 250 mLround bottom flask. The solution was stirred for about 18 hrs at whichpoint the derivatized polyhydric polymer had dissolved. The flask wasthen purged with nitrogen. 0.191 g 3-Mercaptopropionoic acid (MPA) wasthen added to the derivatized HA solution. The pH of the reactionmixture was adjusted to about 9 using 0.25 M NaOH. The solution wasstirred for 4 hours after which the pH was adjusted to about 7 using0.25 M HCl. About 1.8 g NaCl was added to the reaction solution. Thesolution was stirred until the NaCl had dissolved. 200 mL cold acetonewas slowly added to the solution. The reaction mixture was stirred for1.5 hours. 50 mL ethanol was added and the mixture was stirred for 15minutes. The precipitate was isolated using vacuum filtration. Theprecipitate was washed 4 times with 25 mL ethanol in such a manner thatthe filter funnel did not run dry. The precipitate was dried undervacuum at room temperature. A sample of the derivatized polyhydricpolymer was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thepresence of MPA and MSA substitution was evidenced by peaks at 2.1 to3.2 ppm.

Example 53 DVS Modified HA—HA-DVS-37

11.33 g sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) was added to aglass 5 L reaction kettle. The lid, overhead stirrer and anchor impellorwere attached to the reaction kettle. 1133 g deionized water was addedto the kettle. The temperature controller for the Bioreactor heater(Chemglass CLS-1380-19V) was set to 25° C. The solution was stirred atabout 300 rpm for approximately 18 hrs. The stirring speed was increasedto 750 rpm. 35 g of a 1M NaOH solution was then added to the dissolvedsodium hyaluronate. The pH of the solution was measured after 2 min andwas found to be 12.5. The ph was adjusted to 12.32 using 1M HClsolution. A freshly prepared solution of 50 g divinyl sulfone in 282.5 gof DI water was then rapidly added to the stirring solution. The pH wasmonitored and adjusted with 1M NaOH to maintain pH range of 12.2-12.3over the course of the reaction time of ten (10) minutes. After 10minutes, 35 g of a 1M HCl solution was added to the reaction mixture andthe pH of the reaction mixture was adjusted to a value between 5 and 7.About 19.5 g NaCl was then added to the solution. Once the NaCl haddissolved, 2 L acetone was slowly added over a period of <30 minutes.The suspension was stirred for about 3 hours. 400 mL ethanol was addedand the solution was stirred for about 30 minutes. The precipitate wasfiltered under vacuum using a sintered glass funnel. Once all thesolution had been filtered, the vacuum was disconnected and 200 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 timeswith each aliquot standing in static ethanol for five (5) minutes priorto applying vacuum. The product was used for reactions described below.The percent substitution was found to be 31% by the following NMRmethod. Approx. 10-20 mg of the dried sample was added to a vial. D₂Owas added to the sample to make the final concentration of the solutionabout 6 mg/mL. The sample was shaken on an orbital shaker untildissolved. Once dissolved, the sample was transferred into a NMR tubeand the ¹H-NMR spectrum of the sample was recorded on a NMRspectrometer. The spectrum was printed out with the specific peaks inthe 6.0-6.4 ppm (2 peaks from the 2 CH₂=protons from the vinyl sulfoneresidue), the 6.6-7.0 ppm (CH peak of vinyl group) and 1.7-2.0 ppm(singlet from the 3 CH3 protons from the N-acetyl group of the HA)regions being integrated. The percent modification is calculated onmolar ratio of the vinyl CH protons (6.8-7 ppm) to the acetamide(1.7-2.0 ppm) protons.

Example 54 HA-DVS Reaction with mercaptosuccinic Acid (MSA)(HA-DVS-37-MSA)

The vinyl sulfone derivatized HA from Example 53 was added to 1200 g DIwater in a 5 L reaction kettle. The temperature controller for theBioreactor heater (Chemglass CLS-1380-19V) was set to 25° C. Thesolution was stirred for about 18 hrs at 300 rpm at which point thematerial had dissolved. The stirring speed was then increased to 500rpm. 2.696 g mercaptosuccinic acid (MSA) was then added to thederivatized HA solution and allowed to stir for ten (10) minutes. The pHof the reaction mixture was adjusted to about 9 using 1 M NaOH. Thesolution was stirred for 4 hours after which the pH was adjusted toabout 7 using 1M HCl. About 20.64 g NaCl was added to the reactionsolution. The solution was stirred until the NaCl had dissolved. 2 Lcold acetone was slowly added to the solution. The reaction mixture wasstirred for 1.5 hours. 400 mL ethanol was added and the mixture wasstirred for 15 minutes. The precipitate was isolated using vacuumfiltration. Once all the solution had been filtered, the vacuum wasdisconnected and 200 mL ethanol was used to rinse the precipitate. Theethanol was then removed by vacuum filtration. This process was repeatedan additional 3 times with each aliquot standing in static ethanol forfive (5) minutes prior to applying vacuum. The product was dried undervacuum at room temp conditions. A sample of the material was dissolvedin D₂O and the ¹H-NMR spectrum was measured. The presence of MSAsubstitution was evidenced by peaks at 2.3 to 3.1 ppm. The MSA molarsubstitution, as calculated from the integrals at 3.0 ppm and 1.7-2 ppm(HA-acetamide) was 20.6%. Rheology results for a 2% (w/v) solution wereas following for flow viscosity (0.1-1000 1/s) and frequency testing(1-10 Hz). See Tables 5 and 6, and FIG. 5.

TABLE 5 Shear Rate HA-DVS-37-3-MSA (1/s) Average (cP) 0.1 9510 1.15 8063105 1136 1,000 215

TABLE 6 HA-DVS-37-3-MSA Storage Modulus Loss Modulus Frequency AverageAverage 10.00 132.33 84.67 4.96 78.87 69.13 2.46 47.30 53.53 1.00 22.5735.00

Example 55 HA-DVS Reaction with Thiophenol (HA-DVS-37-THIO)

The vinyl sulfone derivatized HA reaction product (produced in the samemanner as Example 53) was added to 660 g DI water in a 5 L reactionkettle. The solution was stirred for about 1 hr at 300 rpm at 30° C.426.06 g ethanol was then added and the solution was stirred for about18 hrs at 300 rpm at 30° C. The stirring speed was then increased to 500rpm. 5.935 g Thiophenol was then added to the derivatized HA solutionand allowed to stir for ten (10) minutes. The pH of the reaction mixturewas monitored and adjusted to about 9 using 1 M NaOH. The solution wasstirred for 2 hours after which the pH was adjusted to about 7 using 1MHCl. About 9 g NaCl was added to the reaction solution. The solution wasstirred until the NaCl had dissolved. 1 L cold acetone was slowly addedto the solution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. Once all the solutionhad been filtered, the vacuum was disconnected and 200 mL ethanol wasused to rinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times with eachaliquot standing in static ethanol for five (5) minutes prior toapplying vacuum. The product was dried under vacuum at room tempconditions. A sample of the material was dissolved in D₂O and the ¹H-NMRspectrum was measured. The thiophenol peaks were observed in the 7.2-7.5ppm. The thiophenol molar substitution was calculated at 32.8%. Rheologyresults for a 2% (w/v) solution were as following for flow viscosity(0.1-1000 1/s) and frequency testing (1-10 Hz). See Tables 7 and 8 andFIG. 6.

TABLE 7 Shear Rate HA-DVS-37-2-THIO (1/s) Average (cP) 0.1 4003 1.153443 105 682 1,000 145

TABLE 8 HA-DVS-37-2-THIO Storage Modulus Loss Modulus Frequency AverageAverage 10.00 99.57 64.00 4.96 55.63 51.27 2.46 31.70 38.70 1.00 14.2024.40

Example 56 HA-DVS Reaction with mercaptobenzoic acid (MBA)(HA-DVS-37-MBA)

The vinyl sulfone derivatized HA reaction (produced in the same manneras Example 53) was added to 660 g DI water in a 5 L reaction kettle. Thesolution was stirred for about 1 hr at 300 rpm at 30° C. 426.06 gEthanol was then added and the solution was stirred for about 18 hrs at300 rpm at 30° C. The stirring speed was then increased to 500 rpm.8.305 g MBA was then added to the derivatized HA solution and allowed tostir for ten (10) minutes. The pH of the reaction mixture was monitoredand adjusted to about 9 using 1 M NaOH. The solution was stirred for 2hours after which the pH was adjusted to about 7 using 1M HCl. About 9 gNaCl was added to the reaction solution. The solution was stirred untilthe NaCl had dissolved. 1 L cold acetone was slowly added to thesolution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. Once all the solutionhad been filtered, the vacuum was disconnected and 200 mL ethanol wasused to rinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times with eachaliquot standing in static ethanol for five (5) minutes prior toapplying vacuum. The product was dried under vacuum at room tempconditions. A sample of the material was dissolved in D₂O and the ¹H-NMRspectrum was measured. The presence of MBA substitution was evidenced bypeaks at 7.1-7.5 ppm (Ar—H). The MBA molar substitution, as calculatedfrom the integrals at 7.1-7.5 ppm (Ar—H) and 1.7-2 ppm (HA-acetamide),was 23.3%. Rheology results for a 2% (w/v) solution were as followingfor flow viscosity (0.1-1000 1/s) and frequency testing (1-10 Hz). SeeTables 9 and 10, and FIG. 7.

TABLE 9 Shear Rate HA-DVS-37-4-MBA (1/s) Average (cP) 0.1 9420 1.15 8167105 1237 1,000 230

TABLE 10 HA-DVS-37-4-MBA Storage Modulus Loss Modulus Frequency AverageAverage 10.00 123.00 94.23 4.96 76.70 76.70 2.46 46.20 58.93 1.00 21.3737.87

Example 57 HA-DVS Reaction with 3-mercapto-1-propanesulfonate(HA-DVS-37-SMPS)

Five (5) grams of vinyl sulfone derivatized HA (produced in the samemanner as Example 53) was added to 500 g DI water in a 5 L reactionkettle. The temperature controller for the Bioreactor heater (ChemglassCLS-1380-19V) was set to 25° C. The solution was stirred for about 18hrs at 300 rpm at which point the material had dissolved. The stirringspeed was then increased to 500 rpm. 1.244 g3-mercapto-1-propanesulfonate) was then added to the derivatized HAsolution and allowed to stir for ten (10) minutes. The pH of thereaction mixture was adjusted to about 9 using 1 M NaOH. The solutionwas stirred for 4 hours after which the pH was adjusted to about 7 using1M HCl. About 8.6 g NaCl was added to the reaction solution. Thesolution was stirred until the NaCl had dissolved. 750 ml cold acetonewas slowly added to the solution. The reaction mixture was stirred for1.5 hours. 150 mL ethanol was added and the mixture was stirred for 15minutes. The precipitate was isolated using vacuum filtration. Once allthe solution had been filtered, the vacuum was disconnected and 100 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 timeswith each aliquot standing in static ethanol for five (5) minutes priorto applying vacuum. The product was dried under vacuum at room tempconditions. A sample of the material was dissolved in D₂O and the ¹H-NMRspectrum was measured. The presence of SMPS substitution was evidencedby peaks at 2.0-2.1 ppm (—CH₂—), 2.5-2.7 ppm (—CH₂—S—) and 2.8-3.0 ppm(—S—CH₂—). The SMPS substitution, as calculated from the integrals at2.5-2.7 ppm (SMPS-CH₂—S—) and 1.7-2 ppm (HA-acetamide), was 26.18%.

Example 58 DVS Modified HA—HA-DVS-36

11.33 g sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) was added to aglass 5 L reaction kettle. The lid, overhead stirrer and anchor impellorwere attached to the reaction kettle. 1133 g deionized water was addedto the kettle. The temperature controller for the Bioreactor heater(Chemglass CLS-1380-19V) was set to 25° C. The solution was stirred atabout 300 rpm for approximately 18 hrs. The stirring speed was increasedto 750 rpm. 35 g of a 1M NaOH solution was then added to the dissolvedsodium hyaluronate. The pH of the solution was measured after 2 min andwas found to be 12.5. The ph was adjusted to 12.32 using 1M HClsolution. A freshly prepared solution of 50 g divinyl sulfone in 282.5 gof DI water was then rapidly added to the stirring solution. The pH wasmonitored and adjusted with 1M NaOH to maintain pH range of 12.2-12.3over the course of the reaction time of twenty (20) minutes. After 20minutes, 35 g of a 1M HCl solution was added to the reaction mixture andpH was adjusted to a value between 5 and 7. About 19.5 g NaCl was thenadded to the solution. Once the NaCl had dissolved, 2 L acetone wasslowly added over a period of <30 minutes. The suspension was stirredfor about 3 hours. 400 mL ethanol was added and the solution was stirredfor about 30 minutes. The precipitate was filtered under vacuum using asintered glass funnel. Once all the solution had been filtered, thevacuum was disconnected and 200 mL ethanol was used to rinse theprecipitate. The ethanol was then removed by vacuum filtration. Thisprocess was repeated an additional 3 times with each aliquot standing instatic ethanol for five (5) minutes prior to applying vacuum. Theproduct was immediately dissolved for the reaction described in Example59. The percent substitution was found to be 51.6% by the following NMRmethod. Approx. 10-20 mg of the dried sample was added to a vial. D20was added to the sample to make the final concentration of the solutionabout 6 mg/mL. The sample was shaken on an orbital shaker untildissolved. Once dissolved, the sample was transferred into a NMR tubeand the ¹H-NMR spectrum of the sample was recorded on a NMRspectrometer. The spectrum was printed out with the specific peaks inthe 6.0-6.4 ppm (2 peaks from the 2 CH₂=protons from the vinyl sulfoneresidue), the 6.6-7.0 ppm (CH peak of vinyl group) and 1.7-2.0 ppm(singlet from the 3 CH₃ protons from the N-acetyl group of the HA)regions being integrated. The percent modification is calculated onmolar ratio of the vinyl CH protons (6.8-7.0 ppm) to the acetamide(1.7-2.0 ppm) protons.

Example 59 HA-DVS Reaction with mercaptosuccinic acid (MSA)(HA-DVS-36-MSA)

The vinyl sulfone derivatized HA from Example 58 was added to 1200 g DIwater in a 5 L reaction kettle. The temperature controller for theBioreactor heater (Chemglass CLS-1380-19V) was set to 25° C. Thesolution was stirred for about 18 hrs at 300 rpm at which point thematerial had dissolved. The stirring speed was then increased to 500rpm. 4.953 g mercaptosuccinic acid (MSA) was then added to thederivatized HA solution and allowed to stir for ten (10) minutes. The pHof the reaction mixture was adjusted to about 9 using 1 M NaOH. Thesolution was stirred for 4 hours after which the pH was adjusted toabout 7 using 1M HCl. About 20.64 g NaCl was added to the reactionsolution. The solution was stirred until the NaCl had dissolved. 2 Lcold acetone was slowly added to the solution. The reaction mixture wasstirred for 1.5 hours. 400 mL ethanol was added and the mixture wasstirred for 15 minutes. The precipitate was isolated using vacuumfiltration. Once all the solution had been filtered, the vacuum wasdisconnected and 200 mL ethanol was used to rinse the precipitate. Theethanol was then removed by vacuum filtration. This process was repeatedan additional 3 times with each aliquot standing in static ethanol forfive (5) minutes prior to applying vacuum. The product was dried undervacuum at room temp conditions. A sample of the material was dissolvedin D₂O and the ¹H-NMR spectrum was measured. The presence of MSAsubstitution was evidenced by peaks at 2.3 to 3.1 ppm. The MSA molarsubstitution, as calculated from the integrals at 3.0 ppm and 1.7-2 ppm(HA-acetamide) was 33.2%. Rheology results for a 2% (w/v) solution wereas following for flow viscosity (0.1-1000 1/s) and frequency testing(1-10 Hz). See Tables 11 and 12 and FIG. 8.

TABLE 11 Shear Rate HA-DVS-36-MSA (1/s) Average 0.1 10667 1.15 8923 114503 105 1237 1,000 231

TABLE 12 HA-DVS-36-MSA Storage Modulus Loss Modulus Frequency AverageAverage 10.00 115.33 71.50 4.96 65.77 57.87 2.46 38.50 44.40 1.00 18.0728.73

Example 60 HA-DVS Reaction with Thiophenol (HA-DVS-36-THIO)

The vinyl sulfone derivatized HA reaction product (produced in the samemanner as Example 58) was added to 660 g DI water in a 5 L reactionkettle. The solution was stirred for about 1 hr at 300 rpm at 30° C.426.06 g Ethanol was then added and the solution was stirred for about18 hrs at 300 rpm at 30° C. The stirring speed was then increased to 500rpm. 10.881 g Thiophenol was then added to the derivatized HA solutionand allowed to stir for ten (10) minutes. The pH of the reaction mixturewas monitored and adjusted to about 9 using 1 M NaOH. The solution wasstirred for 2 hours after which the pH was adjusted to about 7 using 1MHCl. About 9 g NaCl was added to the reaction solution. The solution wasstirred until the NaCl had dissolved. 1 L cold acetone was slowly addedto the solution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. Once all the solutionhad been filtered, the vacuum was disconnected and 200 mL ethanol wasused to rinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times with eachaliquot standing in static ethanol for five (5) minutes prior toapplying vacuum. The product was dried under vacuum at room tempconditions. A sample of the material was dissolved in D₂O and the ¹H-NMRspectrum was measured. The thiophenol peaks were observed in the 7.2-7.5ppm. The thiophenol molar substitution was calculated at 50%. Rheologyresults for a 2% (w/v) solution were as following for flow viscosity(0.1-1000 1/s) and frequency testing (1-10 Hz). See Tables 13 and 14,and FIG. 9.

TABLE 13 Shear Rate HA-DVS-36-thio (1/s) Average (cP) 0.1 8880 1.15 746711 3850 105 1090 1,000 209

TABLE 14 HA-DVS-36-Thio Storage Modulus Loss Modulus Frequency AverageAverage 10.00 97.27 66.13 4.96 56.70 52.80 2.46 33.10 39.97 1.00 15.4025.43

Example 61 HA-DVS Reaction with Mercaptobenzoic Acid (MBA)(HA-DVS-36-MBA)

The vinyl sulfone derivatized HA reaction product (produced in the samemanner as Example 58) was added to 660 g DI water in a 5 L reactionkettle. The solution was stirred for about 1 hr at 300 rpm at 30° C.426.06 g ethanol was then added and the solution was stirred for about18 hrs at 300 rpm at 30° C. The stirring speed was then increased to 500rpm. 15.227 g MBA was then added to the derivatized HA solution andallowed to stir for ten (10) minutes. The pH of the reaction mixture wasmonitored and adjusted to about 9 using 1 M NaOH. The solution wasstirred for 2 hours after which the pH was adjusted to about 7 using 1MHCl. About 9 g NaCl was added to the reaction solution. The solution wasstirred until the NaCl had dissolved. 1 L cold acetone was slowly addedto the solution. The reaction mixture was stirred for 1.5 hours. Theprecipitate was isolated using vacuum filtration. Once all the solutionhad been filtered, the vacuum was disconnected and 200 mL ethanol wasused to rinse the precipitate. The ethanol was then removed by vacuumfiltration. This process was repeated an additional 3 times with eachaliquot standing in static ethanol for five (5) minutes prior toapplying vacuum. The product was dried under vacuum at room tempconditions. A sample of the material was dissolved in D₂O and the ¹H-NMRspectrum was measured. The presence of MBA substitution was evidenced bypeaks at 7.1-7.5 ppm (Ar—H). The MBA molar substitution, as calculatedfrom the integrals at 7.1-7.5 ppm (Ar—H) and 1.7-2 ppm (HA-acetamide),was 52.5%. Rheology results for a 2% (w/v) solution were as followingfor flow viscosity (0.1-1000 1/s) and frequency testing (1-10 Hz). SeeTable 15 and FIG. 10.

TABLE 15 HA-DVS-36-5-MBA Storage Modulus Loss Modulus Frequency AverageAverage 10.00 116.00 77.50 4.96 67.67 63.03 2.46 40.07 48.53 1.00 18.9031.47

Example 62 HA-DVS Reaction with 3-mercapto-1-propanesulfonate(HA-DVS-36-SMPS)

The vinyl sulfone derivatized HA product (produced in the same manner asexample 58) was added to 1200 g DI water in a 5 L reaction kettle. Thetemperature controller for the Bioreactor heater (ChemglassCLS-1380-19V) was set to 25° C. The solution was stirred for about 18hrs at 300 rpm at which point the material had dissolved. The stirringspeed was then increased to 500 rpm. 6.40 g3-mercapto-1-propanesulfonate) was then added to the derivatized HAsolution and allowed to stir for ten (10) minutes. The pH of thereaction mixture was adjusted to about 9 using 1 M NaOH. The solutionwas stirred for 4 hours after which the pH was adjusted to about 7 using1M HCl. About 20.64 g NaCl was added to the reaction solution. Thesolution was stirred until the NaCl had dissolved. 2 L cold acetone wasslowly added to the solution. The reaction mixture was stirred for 1.5hours. 400 mL ethanol was added and the mixture was stirred for 15minutes. The precipitate was isolated using vacuum filtration. Once allthe solution had been filtered, the vacuum was disconnected and 200 mLethanol was used to rinse the precipitate. The ethanol was then removedby vacuum filtration. This process was repeated an additional 3 timeswith each aliquot standing in static ethanol for five (5) minutes priorto applying vacuum. The product was dried under vacuum at room tempconditions. A sample of the material was dissolved in D₂O and the ¹H-NMRspectrum was measured. The presence of SMPS substitution was evidencedby peaks at 2.0-2.1 ppm (—CH₂—), 2.5-2.7 ppm (—CH₂—S—) and 2.8-3.0 ppm(—S—CH₂—). The SMPS substitution, as calculated from the integrals at2.5-2.7 ppm (SMPS-CH₂—S—) and 1.7-2 ppm (HA-acetamide), was 54.3%.Rheology results for a 2% (w/v) solution were as following for flowviscosity (0.1-1000 1/s) and frequency testing (1-10 Hz). See Tables 16and 17 and FIG. 11.

TABLE 16 Shear Rate HA-DVS-36-SMPS (1/s) Average (cP) 0.1 9673 1.15 743311 3443 105 951 1,000 190

TABLE 17 HA-DVS-36-SMPS Storage Modulus Loss Modulus Frequency AverageAverage 10.00 126.13 76.53 4.96 77.77 63.73 2.46 49.50 50.63 1.00 26.4334.73

Example 63 DVS Modified HA—HA-DVS-37 L Lot 1 with Lower MW SodiumHyaluronate

11.33 g sodium hyaluronate (0.62 m3/kg) was added to a glass 5 Lreaction kettle. The lid, overhead stirrer and anchor impellor wereattached to the reaction kettle. 1133 g deionized water was added to thekettle. The temperature controller for the Bioreactor heater (ChemglassCLS-1380-19V) was set to 25° C. The solution was stirred at about 300rpm for approximately 18 hrs. The stirring speed was increased to 750rpm. 30 g of a 1M NaOH solution was then added to the dissolved sodiumhyaluronate. The pH of the solution was measured after 2 min and wasfound to be 12.26. The ph was adjusted to 12.30 using 1M HCl solution. Afreshly prepared solution of 50 g divinyl sulfone in 282.5 g of DI waterwas then rapidly added to the stirring solution. The pH was monitoredand adjusted with 1M NaOH to maintain pH range of 12.2-12.3 over thecourse of the reaction time of ten (10) minutes. After 10 minutes, 27 gof a 1M HCl solution was added to the reaction mixture and pH wasadjusted to a value between 5 and 7. About 19.5 g NaCl was then added tothe solution. Once the NaCl had dissolved, 2 L acetone was slowly addedover a period of <30 minutes. The suspension was stirred for about 3hours. 500 mL ethanol was added and the solution was stirred for about30 minutes. An additional 500 ml of cold acetone was added to ensureeverything had precipitated. The precipitate was filtered under vacuumusing a sintered glass funnel through a 0.22 μm PTFE filter membrane.Once all the solution had been filtered, the vacuum was disconnected and200 mL ethanol was used to rinse the precipitate. The ethanol was thenremoved by vacuum filtration. This process was repeated an additional 3times with each aliquot standing in static ethanol for five (5) minutesprior to applying vacuum. The product was immediately dissolved for thereaction described in Example 64. The percent substitution, asdetermined by the following NMR method, was found to be 25.1%. Approx.10-20 mg of the dried sample was added to a vial. D₂O was added to thesample to make the final concentration of the solution about 6 mg/mL.The sample was shaken on an orbital shaker until dissolved. Oncedissolved, the sample was transferred into a NMR tube and the ¹H-NMRspectrum of the sample was recorded on a NMR spectrometer. The spectrumwas printed out with the specific peaks in the 6.0-6.4 ppm (2 peaks fromthe 2 CH₂=protons from the vinyl sulfone residue), the 6.6-7.0 ppm (CHpeak of vinyl group) and 1.7-2.0 ppm (singlet from the 3 CH₃ protonsfrom the N-acetyl group of the HA) regions being integrated. The percentmodification is calculated on molar ratio of the vinyl CH protons (6.8-7ppm) to the acetamide (1.7-2.0 ppm) protons.

Example 64 HA-DVS Reaction with Thiophenol (HA-DVS-37 L-THIO)

The vinyl sulfone derivatized HA reaction product described in Example63 was added to 660 g DI water in a 5 L reaction kettle. The solutionwas stirred for about 1 hr at 300 rpm at 30° C. 426.06 g ethanol wasthen added and the solution was stirred for about 18 hrs at 300 rpm at30° C. The stirring speed was then increased to 500 rpm. 5.935 gThiophenol was then added to the derivatized HA solution and allowed tostir for ten (10) minutes. The pH of the reaction mixture was monitoredand adjusted to about 9 using 1 M NaOH. The solution was stirred for 2hours after which the pH was adjusted to about 7 using 1M HCl. About 9 gNaCl was added to the reaction solution and stirred until the NaCl haddissolved. 1 L cold acetone was slowly added to the solution. Thereaction mixture was stirred for 1.5 hours. The precipitate was isolatedusing vacuum filtration. Once all the solution had been filtered, thevacuum was disconnected and 200 mL ethanol was used to rinse theprecipitate. The ethanol was then removed by vacuum filtration. Thisprocess was repeated an additional 3 times with each aliquot standing instatic ethanol for five (5) minutes prior to applying vacuum. Theproduct was dried under vacuum at room temp conditions. A sample of thematerial was dissolved in D₂O and the ¹H-NMR spectrum was measured. Thethiophenol peaks were observed in the 7.2-7.5 ppm. The thiophenol molarsubstitution was calculated at 24.4%. Rheology results for a 2% (w/v)solution were as following for flow viscosity (0.1-1000 1/s) andfrequency testing (1-10 Hz). See Tables 18 and 19 and FIG. 12.

TABLE 18 Shear Rate HA-DVS-37L-Thio (1/s) Average (cP) 0.1 1154 1.15 21811 200 1,000 123

TABLE 19 HA-DVS-37L-thio Storage Modulus Loss Modulus Frequency AverageAverage 10.00 13.50 10.97 4.96 3.33 5.51 2.46 0.84 2.77 1.00 0.14 1.13

Example 65 Concentration Effect on Rheological Properties of SodiumHyaluronate Derivatives

Sodium Hyaluronate (Shiseido 1.4 m3/kg, approx. 800 kDa) and thederivatives described in Examples 54, 55, 56, 60, 61, 62, and 63 weredissolved in deionized H₂O into 2% (w/v), 1% (w/v), and 0.5% (w/v)solutions. Flow curve viscosity (0.1-1000 1/s) testing was performed toproduce the following results.

TABLE 20 Flow Curve Viscosity Values of Derivatives described inExamples 54, 55, and 56 Viscosity (cP) Viscosity (cP) Viscosity (cP)HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS-Frequency 37-MBA 37-MBA 37-MBA 37-Thio 37-Thio 37-Thio 37-MSA 37-MSA37-MSA (1/s) 2% 1% 0.5% 2% 1% 0.5% 2% 1% 0.5% 0.1 9420 807 86 4003 86994 9510 1162 108 1.15 8167 991 176 3443 937 160 8063 1237 195 11 4330775 165 2067 715 146 4063 872 180 105 1237 339 100 682 307 87 1136 352101 1,000 230 89 36 145 81 31 215 90 34

TABLE 21 Flow Curve Viscosity Values of Derivatives described inExamples 59, 60, 61 and 62 Viscosity (cP) Viscosity (cP) HA-DVS- HA-DVS-HA-DVS- HA-DVS- HA-DVS- HA-DVS- Frequency 36-MBA 36-MBA 36-MBA 36-SMPS36-SMPS 36-SMPS (1/s) 2% 1% 0.5% 2% 1% 0.5% 0.1 16667 849 208 9673 1487158 1.15 11893 864 204 7433 1460 272 11 5590 644 169 3443 924 218 1051453 275 95 951 341 108 1,000 262 74 32 190 86 35 Viscosity (cP)Viscosity (cP) HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS- HA-DVS- Frequency36-Thio 36-Thio 36-Thio 36-MSA 36-MSA 36-MSA (1/s) 2% 1% 0.5% 2% 1% 0.5%0.1 8880 782 151 10667 794 175 1.15 7467 813 183 8923 882 205 11 3850617 163 4503 661 178 105 1090 269 94 1237 281 99 1,000 209 73 33 231 7534

REFERENCE TABLE 22 Sodium Hyaluronate (Shiseido 1.4 m3/kg, approx. 800kDa) Frequency Viscosity (cP) (1/s) HA 2% HA 1% HA 0.5% 0.1 11,500 141316 1.15 10,500 1543 242 11 6,110 1113 227 105 1810 433 125 1,000 332 10240

Example 66 Ultraviolet Curing as Crosslinking Mechanism for HyaluronicAcid

Functionalized HA-DVS polymers synthesized in methods disclosed herein(e.g., Example 58) with ^(˜)50% derivatization was diluted to a 2% (w/v)solution in DI water and allowed to dissolve overnight. This solutionwas combined with photoinitiator at a loading percentage of 2.5% witheither 1) 2-Hydroxy-4(2-hydroxyethoxy)-2-methylpropiohenone (Irgacure2959, Sigma-Aldrich Corp., St. Louis, Mo., USA) 2) Irgacure 2959(Sigma-Aldrich Corp., St. Louis, Mo., USA) dissolved in ethanol or 3)Omnirad 380 P1108420 (Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide;Sigma-Aldrich Corp., St. Louis, Mo., USA). Each sample was mixed using amixer, Flacktek DAC 150.1 FVZ-K (Flacktek, Inc, 1708 Hwy. 11 Bldg. G ILandrum, S.C. 29356), with 2 cycles of mixing each of duration 30 sec at2000 rpm. Solutions were clamped between glass plates with 1/32″- 3/32″spaces and placed under ultraviolet light (365 nm longwave UV) for ten(10) minute intervals.

Example 67 Ultraviolet Curing as Crosslinking Mechanism for HyaluronicAcid and Hyaluronic Based Derivatives

Functionalized HA-DVS polymers, were synthesized as disclosed herein,for example, Example 58, with ^(˜)50% derivazation and reacted withmercaptobenzoic acid produced in similar methods to Example 61(HA-DVS-16-MBA) and was diluted to a 4% (w/v) solution in DI water andallowed to dissolve overnight. The dissolved solution was combined inthe following HA-DVS to HA-DVS-MBA volume ratios: 25/75, 50/50 and 75/25and mixed using the Flacktek DAC 150.1 FVZ-K with 2 cycles of mixingeach of duration 30 sec at 2000 rpm. The photoinitiator Irgacure 2959was tested at 2.5% and 7.5% loading for both the 75/25 and 25/75 ratios.The photoiniator TPO-L (Ethyl (2,4,6-trimethylbenzoyl)phenylphosphinate, BASF Corporation, 1609 Biddle Avenue, Wyandotte,Mich. 48192, USA) was tested at 7.5% for the 75/25 and 25/75 ratios.Solutions were clamped between glass plates with 1/32″- 3/32″ spacersand placed under ultraviolet light (365 nm longwave UV) for ten (10)minute intervals. Crosslinked films were produced.

Example 68 Cell Culture of Primary Human Dermal Fibroblasts

Primary human dermal fibroblasts (HDFs) (ATCC PCS-201-012, American TypeCulture Collection, Manassas, Va., US) were cultured in Fibroblast BasalMedium (PCS-201-030) supplemented with Fibroblast growth kit-low serum(ATCC PCS-201-041) and Penicillin-Streptomycin-Amphotericin B solution(ATCC PCS-999-02). The fibroblast growth kit-low serum constituents andfinal concentration were: L-glutamine: (7.5 mM), rh FGF basic (5 ng/mL),rh Insulin (5 μg/mL), Hydrocortisone (1 μg/mL), ascorbic acid (50μg/mL), and fetal bovine serum (2%). HDFs were cultured for a minimum offive (5) days prior to cell passaging/seeding for experimentationpurposes. The cell passaging procedure consisted of rinsing cell flasksfor two (2) times with Dulbeco's phosphate buffered saline (1×, DPBS,ATCC 30-2200). Trypsin/EDTA Solution (ATCC PCS-999-003) was then appliedfor 4-8 minutes incubated at 37° C. until the majority of cells haddislodged from the flask surface. Trypsin Neutralizing Solution (ATCCPCS-999-004) was then applied to the solution and the entire amountcentrifuged for five (5) minutes. The trypsin/neutralizing solution wasthen aspirated and the pellet resuspended in medium. An aliquot of theresuspended cell solution was diluted with Trypan Blue (0.4% solution)(Gibco 15250061) at a ratio of 1:1 and counted using a hemocytometer todetermine the initial cell concentration.

Example 69

Polystyrene cell plates (48 wells) were coated with either 2%, 1% or0.5% solutions of deionized H₂O and Sodium Hyaluronate or the polymerderivatives described in Examples 54, 55, 56, 57, 59, 60, 61, and 62.The plates were allowed to dry for 24-48 hours under laminar flowconditions. The sodium hyaluronate and derivatives as described hereinwere then crosslinked with 2% FeCl3 solution in deionized H₂O for 5-15minutes and rinsed with Dulbecco's Phosphate Buffered Saline DPBS. Theresulting films were allowed to dry under laminar flow conditions for anadditional 12-24 hours. Isopropyl alcohol (IPA) was applied to the filmsand then rinsed with DPBS to remove any residual IPA. Human dermalfibroblasts (HDFs) cultured and passaged as described in Example 68 wereseeded at a concentration of 2.5×104 cells/ml. The HDFs were culturedfor five days after which CellTiter 96° AQueous One Solution CellProliferation Assay (MTS, Promega, Madison Wis.) was applied in order toindirectly measure the metabolic activity the vialble cells. Therelative absorbance level was measured at 490 nm on the BioTek ELx808microplate reader and represented in FIG. 13. Results indicated HDFpresence and cell viability support of sodium hyaluronate derivatives.

Example 70

Co-spun (Poly(lactic-co-glycolic acid) (PLGA) (Purasorb PLG1017) andpolydioxanone (PDO) (Resomer X 206S) electrospun substrates were coatedwith 2% (w/v) solutions with either sodium hyaluronate (Shiseido 1.4m3/kg, approx. 800 kDa, Shiseido Co., Ltd. Frontier Science BusinessDivision,1-6-2 Higashi-shimbashi, Minato-ku, Tokyo 105-8310, Japan) ,sodium hyaluronate derivatized with thiophenol (THIO) as describedherein, or sodium hyaluronate derivatized with mercaptosuccinic acid(MSA), as described herein. The coated electrospun matrices were allowedto dry at atmospheric conditions and then were crosslinked using a 2%(w/v) solution of FeCl3 and deionized H2O. Samples of the electrospunmatrices were placed for five (5) minutes in 2% FeCl3 solution on anorbital shaker and then They were then transferred to DPBS and rinsedtwice for five minutes each prior to drying. Control electrospunmaterial without coating was treated under the same cross-linkingconditions. After drying, the electrospun samples were punched into0.95cm2 samples and rinsed in 100% isopropyl alcohol and then rinsedagain in DPBS and allowed to dry under a sterile laminar hood. Thesamples were then placed within 48 well polystyrene cell plates andhydrated with 100 μl DPBS and seeded with cells at 1×104 cells/ml andincubated at 37° C., 5% CO2 cell culture conditions for seven (7) days.Analysis included CellTiter 96° AQueous One Solution Cell ProliferationAssay (MTS, Promega, Madison Wis.) for both 1) the electrospun substratewhich was transferred to another plate in order to isolate the cellsattached to the electospun substrate and 2) the cells remaining on thepolystyrene surface that had migrated or proliferated onto thepolystyrene well during the seven (7) day incubation period. See FIG.14. Additionally, for the case of the electrospun substrates, phalloidin(phalloidin-tetramethylrhodamine B isothiocyanate) (Sigma AldrichP-1951, St. Louis Mo.) staining for f-actin with DAPI(4′,6-diamidino-2-phenylindole, dihydrochloride) staining confirmed thecells' presence and morphology on the electrospun substrates. See FIG.14. Results indicate that the electrospun materials with the derivatizedmaterials support HDF viability and do not indicate a cytotoxic responseto surrounding HDF cells that may not be attached to the electrospunsubstrate.

Example 71 Biologically Active Agents Release from Sodium HyaluronateCross-Linked Derivatives

Sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) and derivatives of HAcomprising mercaptobenzoic acid, thiophenol, and mercaptosuccinic acid(as disclosed in Examples 59, 60 and 61) were diluted to 4% (w/v)concentrations in deionized water and mixed with mometasone furoate(Sigma Aldrich PHR 1400-500 mg) at 0.5% (w/w) loading. The polymers werecrosslinked using the addition of 2% FeCl₃ solution (crosslinking agent)in deionized water for 1:1 crosslinking of carboxyl groups of sodiumhyaluronate and chloride of FeCl₃, and pH of the resulting gels wasadjusted to 6.5-7.5 using 0.25M NaOH solution. The release study bufferapplied to each sample consisted of 2% (w/v) sodium dodecyl sulfate(SDS) dissolved in phosphate buffered saline (pH 7.4, Sigma P3813). Allsamples were incubated at 37° C. and sampled for testing over 168 hours.For each timepoint, 0.5 ml of the sample was combined with 1 ml MeOH andthen filtered through a 0.45 μm filter prior to analyzing using HPLCwith a C18 column and 25:75 DI water:methanol as mobile phase at 1ml/min flow rate. The chromatogram was collected using a photodiodearray detector at 265 nm and the amount of mometasone furoate releasedwas calculated from integral value of peak at the retention time of 3.3minutes based on calibration performed previously. See Table 23 forresults.

TABLE 23 % MF Released Hours HA MBA Thio MSA 0 0 0 0 0 1 13.80 11.8731.33 29.15 2 17.22 13.09 30.65 30.48 4 18.92 14.38 29.96 31.74 6 19.8614.55 29.07 30.82 24 21.86 15.57 29.55 32.56 48 21.63 15.76 28.08 32.5672 21.89 16.60 29.30 33.16 168 22.16 16.66 28.50 38.27

Example 72 Vancomycin Hydrochloride Release from Sodium HyaluronateCross-Linked Derivatives

Sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) and HA derivativescomprising mercaptosuccinic acid and thiophenol, as described in Example59 and 60 were diluted to 4% (w/v) concentrations in deionized water andmixed with vancomycin hydrochloride (Sigma Aldrich PHR1732) at 10% (w/w)loading. The polymers were crosslinked using the addition of 2% FeCl₃solution (crosslinking agent) in deionized water for 1:1 linking ofcarboxyl groups of sodium hyaluronate and chloride of FeCl₃ and pH ofthe gels was adjusted to 6.5-7.5 using 0.25M NaOH solution. The releasestudy buffer applied to each sample consisted of phosphate bufferedsaline (pH 7.4, Sigma P3813). All samples were incubated at 37° C. andsampled for testing over 24 hours. For each timepoint, 0.25 ml of thesample was combined with 5 ml H₂O and then filtered through a 0.45 μmfilter prior to analyzing using HPLC with a C18 column and 85:15phosphate buffer (pH 3.0):methanol as mobile phase at 0.8 ml/min flowrate. The chromatogram was collected using a photodiode array detectorat 205 nm and the amount of vancomycin hydrochloride released wascalculated from integral value of peak at the retention time of 11.3minutes based on calibration performed previously.

Summary results based on HPLC analysis are presented in Table 24.

TABLE 24 % Vancomycin Hydrochloride Release time (hours) Vancomycin HAMSA Thio 0 0.00 0.00 0.00 0.00 1 98.03 32.83 60.20 75.71 3 95.40 38.4361.37 76.78 6 92.08 39.24 61.10 77.57 24 90.70 63.46 68.03 79.29 9672.11 69.75 65.74 68.00

Example 73 Bupivicaine Release from Sodium Hyaluronate Cross-LinkedDerivatives

Sodium hyaluronate (1.4 m3/kg, approx. 800 kDa) and HA derivativescomprising mercaptosuccinic acid and thiophenol, as described in Example60 and 61, were diluted to 4% (w/v) concentrations in deionized waterand mixed with bupivacaine hydrochloride monohydrate (Sigma AldrichB5274). The polymers were crosslinked using the addition of 2% FeCl₃solution in deionized water for 1:1 linking of carboxyl groups of sodiumhyaluronate and chloride of FeCl3 and pH of the gels was adjusted to6.5-7.5 using 0.25M NaOH solution. The release study buffer applied toeach sample consisted of 2% (w/v) sodium dodecyl sulfate (SDS) dissolvedin Phosphate buffered saline (pH 7.4, Sigma P3813). All samples wereincubated at 37° C. and sampled for testing over 96 hours. For eachtimepoint, 0.25 ml of the sample was combined with 5 ml MeOH and thenfiltered through a 0.45 μm filter prior to analyzing using HPLC with aC18 column and 30:70 phosphate buffer (pH 5.0):methanol as mobile phaseat 0.6 ml/min flow rate. The chromatogram was collected using aphotodiode array detector at 205 nm and the amount of bupivacainereleased was calculated from integral value of peak at the retentiontime of 7.1 minutes based on calibration performed previously. See Table25.

TABLE 25 % Bupivacaine Released time (hours) Bupivacaine HA MBA Thio 00.00 0.00 0.00 0.00 1 72.54 47.28 60.90 71.91 3 70.10 52.89 64.78 74.216 71.43 54.06 67.06 77.13 24 83.46 58.56 74.03 79.73

Example 74

Poly(lactic-co-glycolic acid) (PLGA) (Purasorb PLG1017) andpolydioxanone (PDO) (Resonner X 206S) were dissolved inhexafluoro-2-propanol (HFIP) at 8-9% (w/v) concentration atapproximately 50° C. Following cooling to room temperature, thesolutions were loaded into syringes and connected into an alternatingneedle array of the two polymer solutions. This alternating arrayenabled the co-spinning or the PLGA and PDO as separate fibers that wereintertwined with each other. A rotating drum collector was set to 140RPM and the voltage set to 25 kV for the electrospinning process. Thedistance from the needle to collector was 19.5 cm. The syringe pumpswere run in the range of 10 ml/hour to 20 ml/hour. After an electrospunsheet was produced, it was placed under heated vacuum (40° C.) fordrying purposes.

Example 75

Poly(lactic-co-glycolic acid) (PLGA) (Purasorb PLG1017) andpolydioxanone (PDO) (Resonner X 206S) were dissolved inhexafluoro-2-propanol (HFIP) at 8-9% (w/v) concentration atapproximately 50° C. Polyethylene Glycol (PEG) 35000 (Sigma Aldrich81310) was dissolved in HFIP at 16% (w/v) concentration at roomtemperature conditions (^(˜)25° C.). Following cooling to roomtemperature, all solutions were loaded into syringes and connected intoa sequential needle array of the three polymer solutions such that eachpolymer solution was electrosprayed separately but onto the samerotating drum. A rotating drum collector was set to 140 RPM and thevoltage set to 25 kV for the electrospinning process. The distance fromthe needle to collector was 18.0 cm. The syringe pumps were run in therange of 8 ml/hour to 20 ml/hour. After an electrospun sheet wasproduced, it was placed under heated vacuum (40° C.) for dryingpurposes. The electrospun sheet had separate fibers of PLGA, PDO and PEG35,000 within the same sheet.

Example 76

Co-spun electrospun substrates as described in Example 74 were submersedinto 1%, or 0.5% HA derivative (as described in examples 56 and 61)aqueous solutions and sonicated for 30s increments up to two (2)minutes. The samples were then dried under room temperature/roomhumidity conditions. The primary attributes of interest included therate and uniformity of substrate wetting during and followingsonication.

Example 77

Co-spun electrospun substrates as described in Examples 72 and 73 werecoated with 2% HA or a HA derivative (as described in examples 54-57,59-62) aqueous solutions. The solution was applied to the surface of theelectrospun material and a compressive force was applied to theelectrospun substrates to work the derivative solutions into thesubstrate. Excess HA or HA derivative solution was skimmed from thesurface of the substrate. The substrates were then dried at roomtemperature/room humidity conditions. The primary attribute of interestwas material wettability.

Example 78

HA or a HA derivative (as described in examples 59-61) was dissolved inDI water in the concentrations of 2%, 4%, 8%, or 12% (w/v). Some sampleswere combined with a 2% (w/v) FeCl₃ crosslinking solution. All sampleswere placed within a mold and frozen >12 hours at −80° C. and thendried/lyophilized at room temperature vacuum conditions >24 hours.Attributes of interest included mold conformity following drying andmaterial rehydration behavior when introduced to an aqueous solution.

Example 79

HA or HA derivatives (as described in examples 59-61) were dissolved inDI water in the concentrations of 2%, 4%, 8%, or 12% (w/v). They werethen combined with chitosan lactate (Heppe Medical Chitosan 43003) (1%or 2% (w/v) in DI water) at varying ratios of the mass of HA or HAderivative to the mass of chitosan lactate at ratios of 95/5, 90/10,80/20, or 50/50. Some samples were combined with a 2% (w/v) FeCl₃crosslinking solution. All samples were placed within a mold andfrozen >12 hours at −80° C. and then dried/lyophilized at roomtemperature vacuum conditions >24 hours. Attributes of interest includedmold conformity following drying and material rehydration behavior whenintroduced to an aqueous solution.

Example 80

HA or HA derivative solid forms (as described in examples 54 and 59)were mixed with chitosan lactate (Heppe Medical Chitosan 43003) (1% or2% (w/v) in DI water) at varying ratios of the mass of HA or HADerivative to the mass of chitosan lactate at ratios of 95/5, 90/10,80/20, or 50/50. Some samples were combined with a 2% FeCl₃ crosslinkingsolution. All samples were placed within a mold and frozen >12 hours at−80° C. and then dried/lyophilized at room temperature vacuumconditions >24 hours. Attributes of interest included mold conformityfollowing drying and material rehydration behavior when introduced to anaqueous solution.

All references disclosed herein, including patent references andnon-patent references, are hereby incorporated by reference in theirentirety as if each was incorporated individually.

It is to be understood that the terminology used herein is for thepurpose of describing specific aspects only and is not intended to belimiting. It is further to be understood that unless specificallydefined herein, the terminology used herein is to be given itstraditional meaning as known in the relevant art.

Reference throughout this specification to “one aspect” or “an aspect”and variations thereof means that a particular feature, structure, orcharacteristic described in connection with the aspect is included in atleast one aspect. Thus, the appearances of the phrases “in one aspect”or “in an aspect” in various places throughout this specification arenot necessarily all referring to the same aspect. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more aspects.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, i.e., one or more,unless the content and context clearly dictates otherwise. It shouldalso be noted that the conjunctive terms, “and” and “or” are generallyemployed in the broadest sense to include “and/or” unless the contentand context clearly dictates inclusivity or exclusivity as the case maybe. Thus, the use of the alternative (e.g., “or”) should be understoodto mean either one, both, or any combination thereof of thealternatives. In addition, the composition of “and” and “or” whenrecited herein as “and/or” is intended to encompass an aspect thatincludes all of the associated items or ideas and one or more otheralternative aspects that include fewer than all of the associated itemsor ideas.

Unless the context requires otherwise, throughout the specification andclaims that follow, the word “comprise” and synonyms and variantsthereof such as “have” and “include”, as well as variations thereof suchas “comprises” and “comprising” are to be construed in an open,inclusive sense, e.g., “including, but not limited to.” The term“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps, or to those that do not materially affect the basicand novel characteristics of the claimed disclosure.

Any headings used within this document are only being utilized toexpedite its review by the reader, and should not be construed aslimiting the disclosure or claims in any manner. Thus, the headings andAbstract of the Disclosure provided herein are for convenience only anddo not interpret the scope or meaning of the aspects.

Where a range of values is provided herein, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure.

For example, any concentration range, percentage range, ratio range, orinteger range provided herein is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means ±20% of theindicated range, value, or structure, unless otherwise indicated.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety. Such documents may be incorporated by reference for thepurpose of describing and disclosing, for example, materials andmethodologies described in the publications, which might be used inconnection with the presently described disclosure. The publicationsdiscussed above and throughout the text are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the inventors are notentitled to antedate any referenced publication by virtue of priordisclosure.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the disclosurepertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific aspects disclosed in thespecification and the claims, but should be construed to include allpossible aspects along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

Furthermore, the written description portion of this patent includes allclaims. Furthermore, all claims, including all original claims as wellas all claims from any and all priority documents, are herebyincorporated by reference in their entirety into the written descriptionportion of the specification, and Applicants reserve the right tophysically incorporate into the written description or any other portionof the application, any and all such claims. Thus, for example, under nocircumstances may the patent be interpreted as allegedly not providing awritten description for a claim on the assertion that the precisewording of the claim is not set forth in haec verba in writtendescription portion of the patent.

The claims will be interpreted according to law. However, andnotwithstanding the alleged or perceived ease or difficulty ofinterpreting any claim or portion thereof, under no circumstances mayany adjustment or amendment of a claim or any portion thereof duringprosecution of the application or applications leading to this patent beinterpreted as having forfeited any right to any and all equivalentsthereof that do not form a part of the prior art.

Other nonlimiting aspects are within the following claims. The patentmay not be interpreted to be limited to the specific examples ornonlimiting aspects or methods specifically and/or expressly disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

1. A hyaluronic acid polymer derivative comprising one or more modifiedhydroxyl groups, wherein the hyaluronic acid polymer derivative has theformula: A) HA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y)_(n), where HA is hyaluronicacid, X is S or NH, R¹ is a substituted or unsubstituted C₁-C₂₀aliphatic or aromatic moiety, and Y is one or more of H, a carboxylicacid group or a salt or ester thereof, a hydroxyl group, a sulfonic acidgroup or a salt thereof, or an amine group, and n is the number ofmodified hydroxyl groups where n≥1; or B)(Y—R²—X—CH₂CH₂SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y)_(n), where HAis hyaluronic acid, X is S or NH, R¹ and R² are each a substituted orunsubstituted C₁-C₂₀ aliphatic or aromatic moiety, wherein R¹ and R² aredifferent from each other, Y may be the same or different, and Y is oneor more of H, a carboxylic acid group or a salt or ester thereof, ahydroxyl group, a sulfonic acid group or a salt thereof, or an aminegroup, or an amine group, and n≥1 and m≥1; or C)(CH₂═CH—SO₂CH₂CH₂O)_(m)-HA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y)_(n), where HA ishyaluronic acid, X is S or NH, R¹ is a substituted or unsubstitutedC₁-C₂₀ aliphatic or aromatic moiety Y is one or more of H, a carboxylicacid group or a salt or ester thereof, a hydroxyl group, a sulfonic acidgroup or a salt thereof, or an amine group, and n≥1 and m≥1. 2.(canceled)
 3. (canceled)
 4. (canceled)
 5. A process for making aderivative polymer of claim 1, comprising: a) reacting hydroxyl groupsof hyaluronic acid (HA) polymer, with divinyl sulfone (DVS) to provide afirst HA derivative; and b) reacting the first HA derivative with anucleophile of a formula X′—R¹—Y, or X′—R²—Y, or both to provide asecond HA derivative; wherein R¹ and R² are different and each is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, X′ isa nucleophilic group of SH or NH₂, and Y is the same or different, and Yis one or more of H, a carboxylic acid group or a salt or ester thereof,a hydroxyl group, a sulfonic acid group or a salt thereof, or an aminegroup.
 6. The process of claim 5, further comprising step c)derivatizing the second HA derivative polymer by repeating, one or moretimes, step a) or step a) and step b).
 7. The process of claim 5 whereinthe second HA derivative is HA-(OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y)n (HA-DVS-N),wherein HA is hyaluronic acid, X is S or NH, wherein R¹ is a substitutedor unsubstituted C₁-C₂₀ aliphatic or aromatic moiety; Y is one or moreof H, a carboxylic acid group or a salt or ester thereof, a hydroxylgroup, a sulfonic acid group or a salt thereof, or an amine group, andn≥1.
 8. The process of claim 5 wherein 0.25-50% of the hydroxyl groupspresent on the HA polymer are derivatized to —OCH₂CH₂SO₂CH₂CH₂—X—R¹—Ygroups, wherein X is S or NH; wherein R¹ is a substituted orunsubstituted C₁-C₂₀ aliphatic or aromatic moiety; and Y is one or moreof H, a carboxylic acid group or a salt or ester thereof, a hydroxylgroup, a sulfonic acid group or a salt thereof, or an amine group. 9.The process of claim 5 wherein 0.25-50% of the hydroxyl groups presenton the HA polymer are derivatized to —OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y and—OCH₂CH₂SO₂CH₂CH₂—X—R²—Y groups, wherein R¹ and R² are different, andeach is a substituted or unsubstituted C₁-C₂₀ aliphatic or aromaticmoiety, X is S or NH; and Y is the same or different, and Y is one ormore of H, a carboxylic acid group or a salt or ester thereof, ahydroxyl group, a sulfonic acid group or a salt thereof, or an aminegroup.
 10. The process of claim 5 wherein 0.25-50% of the hydroxylgroups present on the HA polymer are converted to oxyethyl ethenylsulfone groups of the formula —OCH₂CH₂—SO₂CH═CH₂.
 11. The process ofclaim 5 wherein 0.25-50% of the hydroxyl groups present on the HApolymer are converted to oxyethyl ethenyl sulfone groups of the formula—OCH₂CH₂—SO₂CH═CH₂ and —OCH₂CH₂SO₂CH₂CH₂—X—R¹—Y, wherein R¹ is asubstituted or unsubstituted C₁-C₂₀ aliphatic or aromatic moiety, X is Sor NH; and Y is the same or different, and Y is one or more of H, acarboxylic acid group or a salt or ester thereof, a hydroxyl group, asulfonic acid group or a salt thereof, or an amine group.
 12. Theprocess of claim 5 wherein the first HA derivative is an oxyethylethenyl sulfone derivative of hyaluronic acid with the formula:HA-(OCH₂CH₂SO₂CH═CH₂)n (HA-DVS), wherein n≥1.
 13. The process of claim 5further comprising reacting a product of step b) with a crosslinkingagent to provide a crosslinked polymer.
 14. A derivative of hyaluronicacid prepared by the process of claim
 5. 15. A crosslinked polymerprepared by the process of claim
 13. 16. (canceled)
 17. (canceled)
 18. Acomposition comprising a hyaluronic acid polymer derivative of claim 1and at least one of a pharmaceutically acceptable excipient, a syntheticpolymer, thermosreversible polymer, biodegradable polymer, buffer,complexing agent, tonicity modulator, ionic strength modifier, solvent,anti-oxidant, preservative, viscosity modifier, pH modifier, surfactant,emulsifier, phospholipid, stabilizer or porogen.
 19. A compositionaccording to claim 18 further comprising a biologically active agent.20. (canceled)
 21. A method comprising administering to a subject inneed thereof an effective amount of a hyaluronic acid polymer derivativeof claim 1, wherein the amount is effective to achieve at least one oftreating a wound of the subject, filling a void in the subject,relieving joint pain in the subject, preventing surgical adhesions inthe subject, sealing tissue in the subject, treating bacterial vaginosisin the subject, treating an ocular condition of the subject, treatingmucocitis of the subject, and treating an ear infection of the subject.22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled) 26.(canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. A method ofdrug delivery to a subject in need thereof comprising administering tothe subject an effective amount of a composition comprising a hyaluronicacid polymer derivative according to claim 1, and the drug.
 31. A methodof supporting tissue growth in a subject in need thereof comprisingimplanting in the subject a tissue scaffold comprising a hyaluronic acidpolymer derivative according to claim
 1. 32. A medical device comprisinga derivative of hyaluronic acid according to claim
 1. 33. A method foradditive manufacturing comprising producing, with an additivemanufacturing apparatus, an article comprising a derivative ofhyaluronic acid according to claim
 1. 34. A method for producing anelectrospun material or article, comprising producing, with anelectrospinning device, a material or an article comprising a derivativeof hyaluronic acid according to claim 1.